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An 85th-minute strike by Charlie Sheringham saw Gills beaten at Conference South Ebbsfleet on Saturday afternoon.
Sheringham pounced on an attempted backpass by Bradley Dack inside his own 18-yard box to beat substitute keeper Glenn Morris for the only goal.
It was Fleet's fourth pre-season victory over Football League opposition this summer.
Ebbsfleet had the best of the first half without creating anything to trouble Gills keeper Stuart Nelson.
Central midfielders Daryl McMahon and Michael Corcoran had time to pick their passes and dictate play up against the experimental Gills duo of Callum Davies and Devante McKain, while the hosts also looked useful out wide with Anthony Cook.
Gills struggled to make much impact in attack but threatened after 10 minutes with Jermaine McGlashan making room for a fierce drive from Luke Norris' pass that was diverted over by his own team-mate, Cody McDonald.
Gillingham changed all 11 players at the break and looked stronger in midfield.
Aaron Morris played the holding role, with Doug Loft and Jake Hessenthaler in front and Bradley Dack looking to support Danny Kedwell and Antonio German.
Fleet's first clear chance of the game came moments into the second half with Alex Osborn's shot blocked on the line by Kortney Hause after the ball bounced over Joe Martin's head.
Gills livened up with Kedwell playing in Dack for a shot that was well saved by Preston Edwards.
At the other end, the lively Cook bent an effort wide after exchanging passes with Aiden Palmer as Fleet carved out another half-chance moving into the final 15 minutes.
Anthony Acaheampong then sent a free header over from a Cook corner.
The game looked to be heading for a goalless draw, until substitute Sheringham's late strike.
Ebbsfleet: Edwards, Howe (Sessegnon 37mins), Palmer, McMahon (Sanderson 69mins), Acheampong, Huke (Harrington 72mins), Osborn, Corcoran, Godden (Sheringham 63mins), Fraser (Johnson 79mins), Cook. Subs not used: Hall, Clark, Nguede, Mazzone.
Gillingham (first half): Nelson, Hare, Borrowdale, McKain, Egan, Barrett, McGlashan, Davies, Norris, McDonald, Dickenson. Second half: G Morris, Brister, Martin, Hessenthaler, Legge, Hause, Dack, Loft, Kedwell, German, A Morris.