Rooney ended an eight-match goalless run by heading in Ashley Young's first-half corner at the Levi's Stadium, home of the San Francisco 49ers, in Santa Clara, California.
Shortly after making 11 substitutions, United doubled their lead in the International Champions Cup match thanks to Jesse Lingard's shot from inside the box.
Rafinha's late volley gave Barca a glimmer of hope, but Adnan Januzaj hit back instantly to give Louis van Gaal his third win of the club's pre-season tour of the United States.
Luis Suarez endured a frustrating afternoon in the California heat. The former Liverpool striker hit the woodwork twice and was subjected to some rough treatment by Phil Jones.
David de Gea made his first appearance of the tour after overcoming a back injury, but there was no sign of Angel di Maria, who looks to be moving closer to a switch to Paris St Germain.
Jones assured Suarez was made to feel unwelcome early on, clattering into the forward from behind.
A minute later the Uruguayan beat De Gea with a curling free-kick, but luckily for the Real Madrid target, the ball struck a post and bounced to safety.
De Gea was at full stretch moments later to deny Sergi Roberto, who had been played in by Rafinha.
Barcelona were dominating possession, but it was Van Gaal's men who struck first.
With eight minutes on the clock, Young floated a corner into the box and Rooney powered a header past Marc Andre ter Stegen.
Rooney shoved Adriano in the back as Young's cross came in, but referee Baldomero Toledo allowed the goal to stand despite the defender's protest.
United were playing a dangerous game, trying to pass out from the back while Suarez, Pedro and Rafinha lurked on the edge of the box.
They looked more impressive going forward as the first half progressed, though.
Rooney rounded the keeper but could not finish before Ter Stegen tipped Young's fierce drive over the bar.
Suarez ended the half on a frustrating note, hitting the woodwork again from inside the box.
Just into the second half, Toledo had seen enough of Jones' clumsy behaviour and booked him for taking out Pedro.
Memphis Depay then showed his strength, turning his marker and firing a low shot at Ter Stegen as United looked for a second.
Van Gaal made 11 changes just after the hour. Less than three minutes later, United doubled their lead.
Tyler Blackett played a one-two with James Wilson before crossing to the back post where Lingard drove in from 10 yards.
United looked to be coasting to victory, but Rafinha hammered a superb volley past substitute keeper Sam Johnstone in the dying minutes.
Januzaj put the result beyond any doubt soon after, though, when he held off his marker before driving home from inside the box.
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