While the transfer to the MLS side was announced soon after the end of the season, Messi was only officially unveiled as an Inter Miami player in recent days.
However, his debut against Cruz Azul in a Leagues Cup tie - a competition involving MLS and Mexican sides - has been pencilled in for some time, inevitably leading to a packed DRV PNK Stadium in Fort Lauderdale on Friday night.
Owing to his lack of match fitness, the Argentine superstar begun the fixture on the substitutes' bench, watching teammate Robert Taylor put Inter Miami ahead in the first half.
Messi made his bow in the 54th minute, yet Cruz Azul got back on level terms 11 minutes later through an effort from Uriel Antuna.
Heading into added-on time, a penalty shootout looked likely, but Messi was provided with his moment to steal the show with a free kick in the 94th minute.
From a position marginally to the left-hand side, the 36-year-old proceed to find the top corner of the net, sparking jubilant scenes that Inter Miami fans have barely experienced during a dismal MLS season.
Speaking in an on-field interview, Messi said: "What I saw was the goal. I saw the goal. I knew that I had to score.
"It was the last play of the game and I wanted to score so I didn't go to penalties."
The strike could prove to be the difference between qualification and elimination, Inter Miami now effectively only requiring a draw from their fixture against Atlanta United to advance.
That match is due to take place on Tuesday evening, before Cruz Azul and Atlanta play the concluding fixture next weekend.