The Argentine superstar set up the first two goals at Miami's Nu Stadium, both from corners, to secure the reigning champions' home-field advantage in at least their opening two rounds of the playoffs.
Ian Fray, Luis Suarez and Rodrigo De Paul racked up three first-half strikes for Inter Miami before Liel Abada's brace soon after the break sparked a comeback sealed by Rodolfo Aloko's late strike in a game characterized by awful defending at both ends.
A single point proved enough. Miami finished the regular season second on 60, as third-place New England Revolution failed to take advantage with a surprise 1-0 loss at Orlando, finishing on 58.
Messi's men will now face the Columbus Crew to begin the playoffs.
Runaway leaders Nashville broke the all-time MLS points record with a whopping 77 after beating Cincinnati 2-0.
Though he did not get on the scoresheet himself Saturday, Messi's 26 goals this season are still almost certain to seal his second straight golden boot award.
Petar Musa, whose FC Dallas were in action later Saturday, would need to score five to prevent Messi from becoming the first player to win back-to-back MLS Golden Boots.
Charlotte had an early goal ruled out in the Miami clash for handball against Idan Toklomati, who set up Abada after intercepting Casemiro's weak defensive header back to the goalkeeper.
In the 14th minute, Messi's corner fell unexpectedly to Charlotte's Luca de la Torre, who spooned it up for Fray to open the scoring for Miami.
Another Messi corner in the 33rd minute found Suarez wide open at the near post. The Uruguayan glanced in his header for 2-0, with Messi picking up the assist.
De Paul made it three before halftime, given time and space by Charlotte's defense to slam a long-range shot low to the goalkeeper's right and into the side netting.
Early in the second half, Abada pulled back a goal for the visitors, blasting home substitute Tyger Smalls' sharp cut-back pass from Ashley Westwood's incisive aerial through ball.
Minutes later, Messi uncharacteristically steered wide a straightforward header from the edge of the six-yard box.
Abada bagged a second in the 55th minute, stunning a crowd that had been in party mood moments earlier, with a first-time finish after a one-two with Pep Biel, who played a smart backheel return pass.
In the 90th minute, Aloko bagged the equalizer, responding first to the ball as Smalls' shot ricocheted off the post.
A visibly frustrated Messi hit the post in stoppage time before Aloko came inches from bagging an unlikely winner with a thunderous shot into the outside netting.
Elsewhere on 'Decision Day', with all 30 MLS teams in action, the Western Conference champion Vancouver Whitecaps beat Canadian rivals Montreal 2-0 to seal the second overall seed.
That means Vancouver would be the home team if there should be a rematch of last year's MLS Cup final against Inter Miami.
Columbus took advantage of in-form Cincinnati's stumble with a last-gasp 4-3 win at Chicago to finish seventh and avoid the wild card round.