Heidenheim were held 1-1 by eliminated St Gallen but Djurgården progressed directly with a 3-1 win in their delayed game in Sweden against Legia Warsaw, who hung on in seventh to qualify, as did third-placed Fiorentina after a 1-1 draw at Portugal's Vitoria who finished second.
Lugano drew 2-2 with Pafos and Rapid Vienna dispatched Copenhagen 3-0 to also go through, with their opponents getting a second chance in the play-offs. Cercle Brugge also progressed directly following a 1-1 draw with Istanbul Basaksehir who went out.
In the revamped third-tier European competition, the top eight went through to March's last 16 automatically while places nine to 16 are seeded in February's play-offs against 17 to 24. The bottom 12 are eliminated.
Heidenheim, third bottom in the Bundesliga having lost six in a row domestically, were eighth and going straight through after taking the lead thanks to Norman Theuerkauf's brillant volley on the half hour.
But now eliminated St Gallen hit back through Jozo Stanic in the second half to complicate Heidenheim's first ever European adventure as they finished 16th in the last seeded spot, with a play-off against unseeded Gent or Copenhagen due.
In London, Chelsea had already confirmed their passage to the last 16. But they secured top spot after a 5-1 home victory over Ireland's Shamrock Rovers, who must settle for 10th and a seeded play-off spot.
Teenager Marc Guiu's hat-trick helped seal a 100 per cent record.
Chelsea's Kiernan Dewsbury-Hall, who also scored, told TNT Sports: "A lot of people will say we are big favourites and honestly, we probably are, but in football it is not easy to keep winning.
"Six wins out of six is pretty good. This tournament is relieving a lot of pressure for some of the people who play in the Premier League and it's a chance for young academy lads to make their debuts. It is a good vibe at the moment."
Fellow former Champions League entrants Real Betis and Panathinaikos squeezed through as seeded play-off sides following wins over HJK Helsinki and Dinamo Minsk, who both go out.
Poland's Jagiellonia Bialystok, APOEL Nicosia and Olimpija will also be seeded in the play-offs, while Vikingur Reykjavík, Borac of Bosnia and Herzegovina, Slovenia's Celje, Cypriots Omonoia, Norwegians Molde and Serbian side TSC are the other unseeded sides.
Edinburgh's Hearts, Kazakhstan's Astana and Austria's LASK are among the teams to bow out.
The Champions League and Europa League have the same new format this season but have eight league matches - not six like the Conference League - so do not conclude the league phase until January.
Draws are taking place for play-offs, meaning 24th does not necessarily face ninth for example, in order to avoid teams deliberately trying to target play-off opponents in the last league matchdays.
