Orlando came from behind at the Walt Disney World Resort in Florida to grab three points in the Group A clash, in the first MLS outing since the season was suspended in March due to the coronavirus pandemic.
Nani had set up Chris Mueller's equalizer to cancel out Juan Agudelo's opener, before the former Manchester United attacker put away a 97th-minute winner.
It came after players gathered pre-game, kneeling and raising their fists in a show of solidarity with the Black Lives Matter movement.
Inter Miami opened the scoring early in the second half after a fine team move, Agudelo tapping in a Victor Ulloa cross.
Andres Reyes was taken off on a stretcher before the hour-mark after a frightening injury which saw the Inter Miami defender struck on the throat.
Orlando drew level with 20 minutes remaining, Mueller getting on the end of a lovely cross from Nani.
Just as it looked like the teams were set to share the spoils, Nani tucked away a dramatic winner for Orlando.