The Saints begin their campaign at Liverpool next Sunday and were given an excellent work-out by their German guests, a second-half Graziano Pelle own goal proving the difference between the sides.
Saints boss Ronald Koeman seems to have a good idea of his best line-up and Southampton started with the team who beat Brighton 3-1, which featured Lloyd Isgrove and Dusan Tadic flanking Pelle in attack.
And a Tadic corner created their first opportunity, Leverkusen keeper Bernd Leno called into action to block a close-range Maya Yoshida effort.
Leno then pulled off another fine save, flying through the air to prevent a deflected Tadic shot from finding the top corner.
However, the German side took the lead in the 56th minute, in slightly fortuitous circumstances.
A Hakan Calhanoglu header bounced awkwardly and Pelle's attempted headed clearance bounced past a scrambling Artur Boruc.
Southampton brought on Gaston Ramirez and Saphir Taider, who both shot off-target soon after entering the fray.
But it was Leverkusen who went closest after that, Julian Brandt's shot trickling past Boruc and heading towards the net, only for Ryan Bertrand to get back and clear.