Despite another strong showing from youngster Jesse Lingard, who scored to equalise the home side's early opener, the Premier League champions lost out to a late winner from Yoshihito Fujita.
It means that new manager David Moyes has seen his side beaten by both a Singha All-Star XI and the J-League outfit in their preparations for the new campaign, results that have sandwiched a win over an A-League All-Star team.
Moyes fielded a strong starting XI for the game at the Nissan Stadium - including first-team regulars such as Robin van Persie, Patrice Evra and Tom Cleverley - but they still crashed to another loss.
Defensive mistakes cost Moyes' men and Yokohama were ahead after less than 30 seconds through 37-year-old Marquinhos.
The hungry home side seized the ball straight from kick-off, and although Marquinhos' initial powerful shot was well blocked by David de Gea, the Spaniard could do nothing to prevent the veteran Brazilian from converting the rebound.
United responded well to this early setback and were rewarded through Lingard in the 19th minute. The 20-year-old finished well after some impressive work from new signing Wilfried Zaha.
And it did not take long for the visitors to take the lead, as Masakazu Tashiro turned Adnan Januzaj's cross into his own net.
However, Yokohama started the second period strongly and Brazilian defender Fabio broke free to head home four minutes after the re-start, before Fujita fired past De Gea in the 78th minute to earn his side the win.
United will look to get back to winning ways when they face Cerezo Osaka in their next pre-season assignment on Friday.