Ronald Koeman's St Mary's Stadium bow ended in a 1-0 defeat to Bayer Levekusen thanks to an own goal from new signing Graziano Pelle.
Southampton were arguably the better side during their final pre-season friendly on Saturday, but were unable to make their general superiority show and Pelle's 57th-minute own goal settled matters.
Dudan Tasic, another Southampton new boy, impressed and struck the post in the first half as Koeman's men caught the eye before the break.
But Bundesliga side Leverkusen claimed victory in fortuitous fashion, when former Feyenoord man Pelle directed a corner past Artur Boruc and into his own net.
Several new faces were making their first appearances at St Mary's, with Tadic, Ryan Bertrand and Pelle all slotting into Southampton's starting XI.
The early exchanges were fairly uneventful as Leverkusen forward Hakan Calhanoglu's wayward effort was the only chance of note in the first ten minutes, but things soon livened up and Southampton started to threaten.
Koeman's side enjoyed significant spells of possession and went close in the 21st minute, but Maya Yoshida could only head on to the post following Tadic's dangerous corner.
The Serbian was almost presented with a glorious chance of his own a couple of minutes later, but he could not quite reach Nathaniel Clyne's low cross.
The early stages of the second half progressed with little in the way of goalmouth action, but the deadlock was then inadvertently broken by Pelle.
Gaston Ramirez went closest to equalising for Southampton and Saphir Taider, who joined on loan from Inter earlier in the week, looked lively after coming off the bench.
However, the hosts were unable to break Leverkusen down and fell to their only defeat of pre-season.