MILLWALL'S play-off chances are all but over after they lost at Middlesbrough on Saturday and Derby drew at Aston Villa.
Britt Assombalonga put the hosts in front before the break and Jonny Howson added to the lead in the second half.
Millwall knew they needed to win to have a chance of overhauling Derby, and though they can still mathematically catch Gary Rowett's side the Rams have a far superior goal difference.
The Lions are eighth in the table after Preston won at Sheffield United earlier on Saturday.
Neil Harris' side are three points behind Derby in sixth heading into the last league game of the season against Aston Villa next Sunday.
It will be the first time in three years under Harris that the Lions are not involved in a play-off campaign.
Instead, attentions will now turn to the summer and recruitment for next season's campaign.
It was Millwall's first defeat away from home in the league since January 20 and it will be Middlesbrough now looking ahead to the play-offs.
They went ahead when Assombalonga fired home at the near post in the first half before a serious injury to Ben Marshall forced Harris into a change.
The game was held up after Marshall suffered a knee problem, later revealed to be a serious cut.
Tom Elliott came on and Steve Morison went to the right side of midfield as Millwall pressed for a way back into the game.
But it was the hosts who doubled their advantage when Jordan Archer dropped a cross and Howson stabbed the ball home.
The Lions continued to attack to try to rescue something from the game and Shaun Williams headed straight at Darren Randolph with nine minutes left.
Harris sent on Shane Ferguson for his first involvement in a league game since February but Boro coasted to the win that guarantees their place in the play-offs.
Millwall face Villa at home next weekend, but trail Derby by three points. The Rams have a plus-eight better goal difference over Millwall.
Image: Millwall FC