The Clarets’ hopes of advancing in the Premier League Cup suffered a major blow with a second successive defeat in Group G.
Burnley will now have to win their final two games – away at Crystal Palace and group leaders Blackburn Rovers – to have any chance of securing a top-two finish and progress to the knock-out stages.
And with many of their more senior players out on loan that looks a demanding task.
The Clarets had five scholars in their starting line-up against a Boro side which scored either side of the break through Tyrone O’Neill and then survived a late Ali Koiki penalty to tighten their grip on second place with a second group win.
The Teessiders also missed a penalty of their own as Lukas Jensen saved a spot-kick for the second game running as the Dane – fresh from signing a new contract at Turf Moor – did his best to keep the Clarets in the game.
Boro had needed a controversial late penalty in the first meeting between the sides to deny the Clarets an opening win.
But despite Koiki’s 10th goal of the season it was never that close at the Tameside Stadium as Andy Farrell’s side was comfortably second best.
The visitors could have led after 25 minutes when dangerman Stephen Walker was tripped in the box by Kane Patterson on the full-back’s first start at U23 level.
However Jensen, who had also saved a penalty in last Friday’s Development League defeat to Barnsley, again may made a fine stop to keep out the effort from Walker and then pounced on the loose ball with Walker collecting a caution for his efforts to get there first.
The Boro frontman, who has three first-team appearances to his name, was a constant threat and was denied again by Jensen as he raced through on goal.
Jensen also stood firm to keep out a shot from impressive Boro skipper Patrick Reading, but he was powerless to prevent the Premier League Two second tier side from going ahead in first-half stoppage time.
Conner Malley’s shot may not have caused any problems but a deflection off O’Neill gave Jensen no chance as it looped into the top corner.
The Clarets had offered little goal threat of their own, with only a long-range effort from Rob Harker and a superb run by Nejai Tucker which released Will Harris the only semblance of chances.
And any prospect of a fightback all but disappeared six minutes into the second half when Boro doubled their lead.
Again, Jensen made a smart save to keep out a shot from Haydon Hackney but O’Neill was again in the right place to tuck away the rebound.
Boro could have added to their lead but Jensen denied substitute Isaiah Jones at his near post and then prevented Walker getting the goal his performance at merited with an excellent save at the forward’s feet.
That save could have taken on extra importance after the Clarets pulled a goal back five minutes from time as Koiki converted from the spot after Jimmy Dunne had been fouled.
But despite a late corner, the Clarets couldn’t find a way through again to grab the point which would have kept them in the hunt to go through.
Burnley: Jensen; Patterson, Thomas, Dunne, Koiki; Tucker, Goodridge, Woods, Conn-Clarke; Harris, Harker (McGlynn HT). Substitutes: Calderbank-Park, Pruti, Thomson, Fenlon.
Middlesbrough: Brynn; Odusina, Wood (Jones 61), Dale; Dodds, Malley, Hackney, Wearne, Reading (Robinson 61); Walker, O’Neill. Substitutes: Robinson, Burrell, Folarin, Flatters.
Referee: Aaron Jackson.