Tranmere recorded their first win in 13 league games to move four points clear of the League One drop zone.
Matt Spring almost opened for Orient against the run of play when his free-kick hit the post.
But Andy Robinson gave the hosts a deserved lead when he collected David Buchanan's corner and fired home.
Enoch Showunmi converted Danny Holmes's first-half injury-time cross and, despite George Porter going close, Rovers held out for a comfortable win.
The O's have now gone five games without a victory and are six points above the bottom four.
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Tranmere Rovers manager Ronnie Moore told BBC Radio Merseyside:
"We've just got to learn to win and how to keep the ball better in the second half.
"There was tremendous application from them all, but overall our keeper hasn't had saves to make and we just about deserved to win.
"It was great finish from Andy Robinson, he was streets ahead of everybody and absolutely fantastic.
"He's got great ability and looks after the ball, he never gives it away, he's a good leader, winner and competitor."
Leyton Orient manager Russell Slade told BBC London 94.9:
"I saw them bombard us for the first 15 minutes and I thought we came through that, but two situations went the wrong way for us.
"The first was the chance to draw when Matty Spring hit the post, and the second was a handball on their second goal.
"It is a clear handball and how the referee can't give that is beyond me to be honest. That gave them the lift they needed at the right time.
"The devastating thing for us was the second goal. It was avoidable."