The Clarets' hat-trick bid came up short at The Den as they were beaten by a strong Millwall side including former Burnley captain Jason Shackell.
Shackell was one of seven players in the Lions' line-up with first-team experience, including striker Aiden O’Brien and goalkeeper David Martin.
Millwall took the lead with just over 30 minutes gone in their final game of the season through Fred Onyedinma, before former Fulham and Blackburn midfielder Ryan Tunnicliffe doubled the hosts' advantage.
Burnley pulled one back in the 88th minute but Tinashe Chakwana’s strike wasn’t enough to avoid only a second defeat in five games for Michael Duff's in-form side who were looking to make it three wins in a row.
The Clarets started off well and came close through a trialist striker who curled an effort just wide of the post.
Shortly after the home side had their first shot on goal, Onyedinma testing Conor Mitchell from just inside the area.
Jason Shackell is on loan at Millwall from Derby County
And it was Millwall who did break the deadlock soon after, Onyedinma managing to beat Mitchell this time as the winger slotted the ball home from outside the box.
Minutes later the lead was doubled as ex-Norwich full back Harry Toffolo latched onto O’Brien's pass before teeing up Tunnicliffe who poked the ball in from close range.
Duff’s men started positively in the second half and came close through efforts from Chakwana and Mace Goodridge.
Shackell then volleyed an effort over the top with a chance to add a third for Millwall.
But the introduction Ntumba Massanka from the bench gave Burnley fresh impetus as he caused for problems for the Millwall defence and his flick-on to Chakwana teed up the striker for an effort that went just wide.
Chakwana (main picture) did then give Burnley a lifeline when his sweet strike found the bottom corner but they couldn't force an equalsier in their penultimate Professional Development League game of a season that finishes at Sheffield Wednesday on Friday.
Burnley: Mitchell, Clarke, Cropper (Bayode 80), Younger, Wilson, N’Guessan, Koiki, Benson, Goodridge, Chakwana, Trialist (Massanka 75). Subs: Stone, Perkins.