Holders Arsenal must go through the play-offs along with Manchester United after missing out on a top-four finish, as Bayern Munich pinched the final automatic quarter-final berth.
Spanish champions Barcelona finished top of the league phase unbeaten after a 2-0 win away to Paris FC, with Vicky Lopez and Caroline Graham Hansen scoring in the French capital.
Three-time Champions League winners Barcelona have reached the final each of the past five years. They pipped Lyon to first place on goal difference after both clubs ended level on 16 points.
This season's format is similar to the men's competition, with the top four teams in the league phase qualifying for the quarter-finals and clubs placed fifth to 12th going into a play-off round.
Lyon thrashed Atletico Madrid 4-0 with a penalty from captain Wendie Renard and goals from Kadidiatou Diani and Korbin Shrader. Vilde Boe Risa's own goal gave Lyon the lead before Atletico were reduced to 10 players.
Chelsea won 2-1 away to Wolfsburg to take third place. Alexandra Popp opened the scoring for the Germans but headers from Lucy Bronze and Sam Kerr ensured the Blues went through to the quarter-finals.
"It was an objective of ours to be in the top four at the start of the season. We probably got that in a different way to what we expected - dropping points early on, but going on and winning games like tonight," Bronze told UEFA.
Bayern eased to a 3-0 win over Norwegians Valerenga to edge Arsenal and United to fourth place by a point.
Olivia Smith and Beth Mead struck in a 3-0 victory for Arsenal away to Belgian outfit Leuven, with Saar Janssen also turning into her own net.
Arsenal picked up their fourth straight win but it wasn't enough to avoid the play-off round after starting the league phase with back-to-back losses.
"We've grown in this Champions League campaign. We're showing a lot more focus and consistency. We're definitely ready for the next round and whoever we come up against," said Mead.
Jess Park's fine finish lifted United to a 1-0 win at Juventus, who had started the night in the top four. United ended level with Arsenal on 12 points.
The draw for the knockout phase takes place on Thursday.
Arsenal could face an instant rematch with Leuven or play Atletico in a two-legged play-off. United will take on whichever side the Gunners do not.