The mouthwatering final pitted defending champions Barcelona against the record Champions League winners Lyon, who nearly took the lead when Lucy Bronze sent the ball crashing against the frame of her own goal.
Barcelona recovered from that nervy moment to offer their own threat, with Patri Guijarro and Caroline Graham Hansen both threatening in the first period in Bilbao.
With the game goalless at the break, Barcelona's star player Aitana Bonmati moved through the gears to find the all-important opener with the aid of a deflection.
Any doubt over the result were quashed when substitute Alexia Putellas swept the ball into the roof of the net to score for the third time in a Champions League final.
Referee Rebecca Welch blew the full-time whistle to spark scenes of job among the Barcelona contingent as they celebrated a phenomenal quadruple, adding the Champions League trophy to the Liga F title, the Copa de la Reina and the Spanish Super Cup.
As for Lyon, they were ultimately denied a ninth European title, but can still look back on the season with pride after reaching their 11th Champions League final and claiming the Division 1 Feminine crown.
Goalless first half
Barcelona made a purposeful start to proceedings at San Mames, with Graham Hansen teeing up Salma Paralluelo to register an early shot on target.
However, the Catalan giants were nearly the architects of their own downfall when Bronze inadvertently flicked a Lyon corner against her own crossbar.
That momentary scare did little to deter the Barcelona momentum as Jonatan Giraldez's side swiftly resumed their search for the crucial opening goal.
France international Selma Bacha was required to hook the ball off the line after Guijarro's strike bounced off Christiane Endler and Lindsey Horan before creeping towards goal.
As a gripping half drew to a close, Graham Hansen drove into the box, before shifting the ball onto her right foot and firing just wide of Endler's goal to ensure that the two sides went into the break on level terms.
Bonmati grabs crucial opener
After having 15 minutes to recharge their batteries, the French champions attempted to catch Barcelona off guard in the opening stages of the second period, with Wendie Renard fizzing an effort over from Horan's cushioned pass.
Lyon were ultimately made to rue that missed opportunity when Bonmati showcased her talents as the reigning Ballon d'Or holder.
The skilful midfielder created the space to receive Mariona Caldentey's neat pass, before darting into the box to chop the ball onto her left foot and fire a deflected shot past the helpless Endler.
As the contest drifted into the final 15 minutes, Lyon started to up the ante as they desperately searched for an equaliser to send the final to extra time.
Kadidiatou Diani attempted to add to the eight European goals that she has scored this season when she cut inside onto her left foot, only to send a lofted strike harmlessly over Cata Coll's goal.
Substitute Ada Hegerberg then failed to hit the target, nodding the ball over the bar after Bacha's inviting delivery.
Putellas adds second to seal Champions League glory
Lyon's trophy hopes continued to fade until they were finally squashed by Putellas in the fifth minute of stoppage time.
The Barcelona icon rifled a left-footed shot into the roof of the net after latching onto a cutback from fellow substitute Claudia Pina.
As a result of the win, the Blaugrana have become just the third team to win the competition on at least three occasions.
Giraldez will celebrate a second straight Champions League crown before he bids his farewells to take over the reins at Washington Spirit in the United States.
As for Sonia Bompastor, she is also expected to leave her position as Lyon boss to succeed Emma Hayes as Chelsea manager.