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The hosts had to come from behind after a Neil Danns opener for the visitors but Andrew Crofts equalised before Byfield's winner a minute from time.
Danns took advantage of a napping defence to score from close range on 50 minutes although he looked offside.
The goal sparked a period of Colchester dominance during which the Essex club could and should have trebled their lead.
On 54 minutes, Chris Iwelumo headed against the post with the goal at his mercy after a storming run from Kem Izzet down the left.
He crossed to the far post where Greg Halford was waiting to tee up the tall striker.
Two minutes after that Halford should have scored when set up by Sam Stockley but he scuffed his shot horribly allowing Jason Brown to make a comfortable save.
Gills manager Neale Cooper responded by sending on new signing Paul Shields in place of veteran midfielder Andy Hessenthaler.
With Gillingham struggling to make any headway, Cooper sent on another attacking player in Matthew Jarvis.
The home side started to pour forward and equalised with 15 minutes to go as Crofts met a Danny Spiller cross from the right with a diving header that powered past Aidan Davison in the Colchester goal.
Then it was left to the livewire Byfield to settled the match with a crisp shot on the turn from the edge of the area.
There was little in the first half to suggest Colchester would dominate after the break as Gillingham had the better chances.
Dangerman Byfield had a goal disallowed on five minutes for handball and then saw Davison beat away a fierce drive on 23 minutes.
Davison denied him again with eight minutes left in the second half, palming away a curling right footed shot as the Gills poured forward looking for the victory.