It looked like both teams would preserve their unbeaten records after Freddy Eastwood struck to cancel out Gary Roberts' opening goal for the hosts. But substitute Gnanduillet nodded home in the fifth minute of added time to send Paul Cook's side joint-top of the table with Oxford.
Marc Richards had a chance to put the Spireites in front after six minutes but he scuffed a shot wide from a narrow angle after going past goalkeeper Dan Bentley.
Cook's men were controlling the game and Bentley had to push over a 25-yard Jay O'Shea shot in the 27th minute. However, Southend almost scored on the break as half-time approached when Anthony Straker set up Conor Clifford, who had a trial at the Proact Stadium this summer, but his shot was deflected over.
Chesterfield broke through in the 59th minute when Clifford brought down Roberts 25 yards out and the midfielder's free-kick looped off the wall and past Bentley.
The Shrimpers equalised in the 68th minute when former Spireite Kevan Hurst touched on a free-kick for Eastwood to fire low past Tommy Lee from 15 yards.
But Gnanduillet turned in Roberts' corner deep into stoppage time to send the majority of the 5,579 inside the Proact Stadium delirious.