Sparta Prague secured top spot in Group K with a battling 1-0 Europa League win at the Generali Arena against Southampton on Thursday.
The Czech side began the game in pole position and the hosts asserted their dominance in the 11th minute, left-back Costa Nhamoinesu converting an excellent Borek Dockal free-kick.
Southampton failed to create a clear-cut chance of note from open play and Claude Puel's men will now have to beat Hapoel Be'er Sheva or secure a goalless draw in their final group game to guarantee their progress into the next round.
Shane Long's late effort was the closest Southampton came to scoring their first away goal in this season's Europa League, with the Premier League team struggling for creativity.
Virgil van Dijk then planted a header over the crossbar as a frustrating match ended in defeat for the Saints, while David Holoubek's men celebrated winning the group after their fourth straight European home victory.
Sparta have an unassailable five-point lead at the top of Group K, two points ahead of Southampton and Hapoel after the Israeli side came from behind to beat 10-man Inter 3-2.
Southampton had the better of the early exchanges, but the hosts took the lead after 10 minutes.
Dockal's cute free-kick was misjudged by the Saints defence and Costa read the flight of the ball perfectly to connect with a low volley that beat Fraser Forster at his near post.
Sam McQueen's low drive and a Long header tested Tomas Koubek as the visitors initially responded well to falling behind, but Sparta's defence restricted the Saints to speculative efforts.
A wild Maya Yoshida shot from well outside the box shortly after half-time summed up Southampton's frustration when faced with Sparta's determined deep-lying defence.
Pierre-Emile Hojbjerg limped off after 54 minutes to further deepen Saints' problems as Puel introduced Sofiane Boufal and Oriol Romeu in a bid to find a foothold in the game.
Forster pushed away a firm strike from Vyacheslav Karavayev and then had to deal with another dangerous Dockal free-kick that only needed a touch from David Lafata to add a second goal.
Lukas Marecek went close with a curling effort after 65 minutes, then Forster denied Dockal after Jordy Clasie gave away the ball on the edge of his own area.
Puel turned to Charlie Austin as his last change, but it was Long who almost found the equaliser after 77 minutes with a shot that bent narrowly wide of the post.
Southampton captain Van Dijk then sent a free header over the crossbar with the goal at his mercy and it proved a key miss as Southampton failed to find an equaliser.