With a win enough to guarantee progression, Atletico turned in a flat performance against the side lying 10th in the Portuguese table, and a goal in each half from Wilson Eduardo gave Academica victory.
While the result is still unlikely to affect the holders' progress to the knockout stages, their status as group winners is now open to question after Viktoria Plzen joined them at the top on nine points.
The in-form Czechs brushed aside hapless Hapoel Tel Aviv 4-0 two goals from Daniel Kolar and one each for David Stipek and Marek Bakos.
Swiss side Young Boys blew the fate of Group A wide open with a shock 3-2 win over Serie A Udinese in Italy.
Raul Bobadilla opened the scoring for Young Boys in the 27th minute and despite Antonio Di Natale's equaliser, they regained the initiative through Alexander Farnerud and Rapha Nuzzolo.
Diego Fabbrini gave the Italians late hope with an 83rd minute effort but their defeat leaves them stranded at the bottom of the table on four points.
Udinese's loss was good news for Premier League side Liverpool, who were beaten by Lacina Traore's 45th minute strike for Anzhi Makhachkala in Moscow. Anzhi now top the group by a point, with Liverpool and Young Boys tied second.
Fenerbahce all but guaranteed progression from Group C with a 2-0 win over AEL in Turkey. Dirk Kuyt and Moussa Sow scored the goals while AEL had Edwin Ouon sent off late on.
The Turks' hopes were boosted by a 2-2 draw between Marseille and Borussia Monchengladbach, which leaves the two sides locked together on five points.
Mike Hanke put the Germans ahead before goals from Joey Barton - direct from a corner - and Jordan Ayew turned the game in Marseille's favour. Juan Arango rescued a potentially crucial point in the third minute of stoppage time.
Newcastle failed to grab the win they needed to guarantee qualification from Group D but will be happy after fighting back for a 2-2 draw at Club Brugge.
Ivan Trickovski and Jesper Jorgensen gave the Belgians a 2-0 lead inside the first 19 minutes before goals from Vurnon Anita and Shola Amoebi kept Alan Pardew's men on track.
Bordeaux stay second after David Bellion's 16th minute goal proved enough for them to see off Maritimo 1-0 in France.
Steaua Bucharest kept control of Group E with a 2-1 win at Molde, Alexandru Chipciu and Iasmin Latovlevici netting before Daniel Chima hit a late consolation.
Goals from Vedad Ibsevic and Martin Harnik kept Stuttgart's hopes alive with a 2-0 win over Copenhagen in Denmark.
Edison Cavani scored all four as Napoli beat Group F leaders Dnipro Dnipropetrovsk 4-2 in Italy. Artem Fedetskiy and Roman Zozulya replied for the visitors, who remain top.
Also in Group F, AIK gave themselves hope as a Mohamed Bangura goal gave them a 1-0 home win over PSV.
Rubin Kazan and Inter Milan booked their respective places in the knock-out stages after securing victories which guaranteed their progress from Group H.
Vladimir Dyadyun's 19th-minute goal gave the Russians a 1-0 win at Neftchi while two second-half strikes from substitute Rodrigo Palacio helped Inter end Partizan's hopes with a 3-1 triumph in Belgrade.
Fredy Guarin also scored for the visitors, while Nemanja Tomic replied for Partizan in injury-time. The group winners are likely to be determined when Rubin face Inter in Russia in a fortnight's time.
Lyon maintained their 100 per cent record and ensured qualification from Group I after beating last year's runners-up Athletic Bilbao 3-2 in Spain.
Bafetimbi Gomis and Yoann Gourcuff put the visitors 2-0 up at the break and despite Ander Herrera and Aritz Aduriz pulling Bilbao level, Alexandre Lacazette grabbed a 63rd-minute winner.
Also in Group I, Sparta Prague all but assured qualification after a 1-1 draw with Ironi Kiryat Shmona in Israel, Leonard Kweuke cancelling out Darko Tasevski's early goal for the hosts.
Two Libor Kozak goals kept Lazio on top of Group J with a 3-0 win over Panathinaikos at the Stadio Olimpico. Sergio Floccari added a 59t- minute third for Vladimir Petkovic's men.
Also in Group J, a Jermain Defoe hat-trick helped Tottenham take a big stride towards qualification with a 3-1 win over Maribor at White Hart Lane. Robert Beric briefly equalised for the visitors.
Ukrainians Metalist Kharkiv confirmed their progress from Group K with a 3-1 win over Rosenborg. Taison, Cleiton Xavier and Juan Manuel Torres scored for the hosts with Borek Dockal replying for the Norwegians.
And Bayer Leverkusen will join Metalist in the knockout stages after coasting to a 3-0 win over Rapid Vienna with goals from Jens Hegeler, Andre Schurrle and Manuel Friedrich.
Hannover booked their passage from Group L after a dramatic 3-2 win over Helsingborg, who finished with nine men.
A double from Mame Biram Diouf gave the Germans a comfortable advantage before Nikola Djurdjic and Alejandro Bedoya dragged the score back level.
But the Swedes had Peter Larsson and Walid Atta both sent off in the closing minutes before Szabolcs Huszti struck the winner with an injury-time penalty. Meanwhile, group rivals Twente and Levante drew 0-0 in Holland.
Marco Streller scored an 80th-minute winner as Basle kept their Group G hopes alive with a 1-0 win over Videoton, while group leaders Genk needed an injury-time equaliser from Glynor Plet to rescue a 1-1 draw at Sporting Lisbon, who despite being down to 10 men due to the dismissal of Stijn Schaars had led through a Ricky van Wolfswinkel goal.