Colombia beat Venezuela 1-0 while 10-man Peru held on for a 0-0 draw at Paraguay as the long journey of South American qualifying for the 2026 World Cup got under way.
Messi, who led Argentina to their third World Cup triumph in Qatar in December, has yet to confirm whether he will continue with his national side all the way to 2026, but he showed his importance yet again with the breakthrough in what had threatened to be a frustrating night for Lionel Scaloni's team.
Lautaro Martinez won a free-kick in a central area in the 78th minute, just outside the Ecuador penalty area and Messi executed to perfection with his effort floating over the wall and dipping perfectly to beat the stationary Hernan Galindez.
With the goal, Messi drew level with former Barcelona teammate Luis Suarez of Uruguay as the all-time leading scorers in the South American qualifiers, both with 29 goals.
The Albiceleste had dominated possession against an Ecuador side which defended in numbers and limited their attacks to the occasional break on the counter.
The first opening of the game fell to Messi in the 16th minute when he was found on the edge of the box but put his side-foot effort wide of the post.
Just before the break, Lautaro Martinez went closer when he slid in to meet a Rodrigo De Paul and his shot struck the post.
Messi worked himself an opening in the 69th minute but his low shot was weakly struck and was comfortably dealt with by Galindez.
But the 80,000 crowd in the Estadio Monumental were brought to their feet by Messi's goal and from then on it was smooth sailing for Argentina.
De Paul, who was excellent throughout, dribbled across from the right setting up substitute Angel Di Maria but his effort was just wide.
Then De Paul brought a fine save out of Galindez near the end with a close-range blast at the near post.
Pinpoint
A 46th minute header from Rafael Santos Borre, from a pinpoint Jhon Arias cross, was enough to earn three points for Colombia in Barranquilla.
The victory ensured a winning start for new Colombia coach, Argentine Nestor Lorenzo, who was taking charge of his first competitive game.
Colombia failed to qualify for last year's World Cup in Qatar while Venezuela are hoping to earn their first ever appearance in a World Cup finals.
Peru's difficult task in Ciudad del Este became harder after they lost defender Luis Advíncula on the stroke of half-time when he was dismissed for a second yellow card.
But they held on for a point in what was a pulsating, high-tempo encounter.
The qualification process for the expanded 48-team World Cup, to be held in the United States, Canada and Mexico, offers two extra places for South America with six teams qualifying directly.
The seventh out of the 10 nations will head to an inter-continental play-off.
On Friday, five-times World Cup winners Brazil face Bolivia while Uruguay host Chile.