The 1996 cartridge was a launch title for the Nintendo 64 console, and was one of the most influential early 3D platformers.
The auction house said there were "fewer than five" copies in such good condition.
The sale dwarfed another record-breaker, an original Legend of Zelda cartridge, set just two days before.
That original Nintendo Entertainment System (NES) cartridge sold for $870,000 on Friday, briefly making it the world's most expensive game.
Neither game was bought for the rarity of its gameplay experience - Nintendo has rereleased both for its Nintendo Switch console.
The Super Mario 64 cartridge was graded by video game collectable firm Wata at a 9.8 A++ rating - meaning it is both in near-perfect condition and its seal is intact and "like new".
A high rating is not enough to guarantee a high auction price - although it helps. Another copy of Super Mario 64 in the same auction with a 9.6 A++ rating sold for a comparatively affordable $13,200.
But rarer copies of more popular games attract the most bids.
Super Mario 64 was the best-selling game for the Nintendo 64 system, and is widely seen as an important game in video game history. It marked Mario's first move into three dimensions, distinct from his side-scrolling origins.
While many modern gamers feel it has not aged particularly well and is widely thought to have significant camera issues, it is still considered among the best video games ever made. Heritage Auctions, the company behind the sale, was taken by surprise at the huge price it eventually attracted.
Ahead of the auction, much of the attention had been on the rare Zelda cartridge.
The 9.0 A-sealed copy of 1988's The Legend of Zelda was the first game in the series, and has gone on to be one of the best-performing and most critically-acclaimed in Nintendo's entire catalogue.