Thomas Tuchel would have been hoping to build on the victory over Club America at the weekend, the Blues being good value for their 2-1 success in Las Vegas.
However, despite heading into half time with a lead, a last-gasp equaliser and a missed spot kick from Conor Gallagher led to Charlotte prevailing 5-3 in a penalty shootout after a 1-1 draw.
Unlike the team selected for the Club America game, there was more of an unfamiliar look to Tuchel's starting XI as Emerson Palmieri played on the left-hand side of a back three, Callum Hudson-Odoi featured at right wing-back and Michy Batshuayi got the nod down the middle of the attack.
After kickoff had been delayed for over an hour due to a storm, both teams had good opportunities with Mateo Kovacic and Karol Swiderski seeing their respective shots saved.
Nevertheless, it was Chelsea who went ahead on the half-hour mark through Christian Pulisic, the American smartly finishing from close range after Batshuayi's shot had been diverted into his path.
The Premier League giants pushed hard for a second with Batshuayi and Marcos Alonso both having shots saved by Kristijan Kahlina, but Charlotte reached the break trailing by just the one goal.
Tuchel again made sweeping changes to his team, the notable introduction being that of summer arrival of Raheem Sterling, but Levi Colwill got his chance in the back three with Ethan Ampadu being used in the middle of the pitch alongside Gallagher.
While Charlotte started to look more threatening, Mason Mount and Sterling also went close for the Blues, and it appeared that Chelsea had all but sewn up the game as it entered added-on time.
However, a foul from Trevoh Chalobah resulted in a Charlotte penalty which was dispatched by Daniel Rios, an equaliser which provided the patient crowd with the added reward of penalties.
A desperately weak attempt from Gallagher proved to be the difference, the former Crystal Palace loanee sending a tame effort straight at George Marks.
Charlotte scored each of their penalties against Edouard Mendy to secure the MLS team an eye-catching triumph against English opposition.