Messi, 38, needed just seven minutes to reach his milestone, coolly tucking away a low finish to give Miami a 1-0 lead in the regional club competition knockout game.
But Nashville's Cristian Espinoza lashed in a 74th-minute equalizer to make it 1-1 on aggregate, and after holding Miami at bay for the remainder of the game, the visitors from Tennessee advanced to the quarter-finals on the away goals rule.
Miami's exit was a bittersweet ending to what had promised to be a night of celebration after Messi's early opener in what was the club's final game at their Chase Stadium home in Fort Lauderdale.
The strike took Messi's tally to 81 goals for Miami following his move to Major League Soccer in 2023.
The eight-time Ballon D'Or winner previously scored 672 goals for Spanish giants Barcelona, 32 for France's Paris Saint-Germain, and a further 115 international goals for Argentina.
Messi's 900th goal comes some 21 years after his first goal in senior football, when he scored for Barcelona in a 2-0 win over Albacete in 2005 as a fresh-faced 17-year-old.
Messi, widely regarded as the greatest player in football history, still has some way to go before he catches his great rival Cristiano Ronaldo, the 41-year-old Portuguese superstar who is on 965 career goals.
Speaking ahead of Messi's 900th goal, Miami manager and former Argentina and Barcelona team-mate Javier Mascherano described the star's goals total as "insane."
"I haven't contributed much to him scoring goals, neither with assists nor now as a coach," Mascherano said.
"I've been lucky enough to see most, or many, of the goals he's scored, much closer than you all, and that's a privilege.
"The number we're talking about (900) is insane, and that's why Leo is a one of a kind."
Clinical finish
Wednesday's match in Fort Lauderdale carries added significance for Inter Miami, marking a farewell to the team's Chase Stadium.
The venue has served as Inter Miami's home since the club entered Major League Soccer in 2020. The team will move into a new purpose-built 26,700-seat facility next month.
Messi is strongly expected to play in his sixth World Cup later this year, when Argentina defend their title in the 48-team tournament being staged in the United States, Canada and Mexico.
The Argentine star has already extended his Inter Miami contract through to the end of the 2028 season.
Messi's prolific goalscoring has been a hallmark of his career. His 672 goals for Barcelona came in just 778 games - an average of 0.86 per game - helping the Catalan club win 35 trophies before departure to Paris Saint-Germain in 2021.
In France, he scored 32 goals in 75 matches, winning three trophies including two league titles.
In Miami, his 81 goals have come in just 96 appearances, helping the team win the 2023 Leagues Cup and the 2025 MLS Cup.
His 115 international goals have come in 196 matches, including two in the 2022 World Cup final victory over France.
Messi's goals tally leaves him second only to Ronaldo according to most recognised statistical records.
Brazilian legend Pele is often said to have scored more than 1,000 goals but those numbers have been the subject to debate. Most statisticians put Pele's total for Brazil, Santos and New York Cosmos at 762.
