Rooney took advantage of an Orlando giveaway in its attacking half, looked up to see Rowe out of his area and hammered it to the goal line.
D.C. United (8-4-6) snapped a five-game winless stretch - with four draws during the span. Orlando (5-8-3) has just two wins in its last seven games.
Bill Hamid made three saves to get his eighth shutout of the season.
In the first matchup of the season, Rooney set up a goal and scored directly from a free kick to help D.C. United win 2-1 on March 31 in Orlando.
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