These two clubs have not met each other since April 2014, when Colchester ran out 4-1 winners despite Brentford ultimately gaining promotion from League One a few weeks later.
Match preview
The two clubs have gone on very different trajectories within those ensuing eight years, of course, with Colchester languishing in the bottom-half of League Two and Brentford competing in the Premier League.
After a late season surge under the tutelage of Wayne Brown guided the U's away from relegation danger, the East Anglian-based club look like they could be set for another tricky campaign ahead of them.
Three defeats from their opening five league games has not been the start the former Ipswich Town defender would have wanted to make, although he and the fanbase alike would have taken great pleasure from beating their nearest rivals in the first round of this competition to set up a home tie against Brentford.
While remaining in the Football League is the club's priority this season, a cup run is always a welcome distraction for any team, with the opportunity of knocking out a top-flight side certain to motivate any group of players.
After the heady heights of thrashing Manchester United 4-0, Brentford were brought back down to earth with a crushing 3-2 defeat at the hands of their west London rivals Fulham on Saturday.
The Bees magnificently fought back from two goals down to look like claiming a precious away point, but Aleksandar Mitrovic had other ideas with his last-gasp header sending Craven Cottage into ecstasy.
Still, Thomas Frank cannot be too greedy, with four points from his side's opening three games representing a decent building block following comfortably avoiding any relegation danger in their debut Premier League season, while the Danish manager is likely to be targeting a cup run this time around.
His side reached the semi-final of the EFL Cup as a Championship outfit two seasons ago, and he will be demanding his side avoid any embarrassment against a team three divisions below them when they travel to Essex on Tuesday.
Colchester United EFL Cup form:
Colchester United form (all competitions):
Brentford form (all competitions):
Team News
Colchester will remain without club captain Tommy Smith due to a foot injury, while midfielder Emyr Huws is likely to remain out of action until October with an ankle issue.
Brown started a strong team in the previous round away to Ipswich, with Cole Skuse set to return to the starting line-up to provide an experienced head in midfield.
Brentford are without Ethan Pinnock until potentially mid-December with a serious knee injury, while Sergi Canos and Kristoffer Ajer remain doubts with muscle strains.
Frank could hand several fringe players crucial minutes ahead of a long season ahead, with goalkeeper Thomas Strakosha set to make his debut for the club after his summer move from Lazio.
Likewise, Keane Lewis-Potter should start for the Bees for the first time having come off the bench at Fulham on Saturday, with the likes of Frank Onyeka, Mads Sorensen and Halil Dervisoglu all hoping to receive a starting berth, too.
Colchester United possible starting lineup:
Hornby; Tchamedeu, Chambers, Eastman, Kazeem; Marshall-Miranda, Skuse; Hannant, Chilvers, Nouble; Sears
Brentford possible starting lineup:
Strakosha; Roerslev, Jansson, Sorensen, Hickey; Baptiste, Onyeka, Janelt; Ghoddos, Dervisoglu, Lewis-Potter
We say: Colchester United 1-2 Brentford
Should Brentford heavily rotate, Colchester will fancy their chances of causing another upset in this competition.
However, the Bees have added to their squad well this summer and should have the firepower to edge past their League Two opponents, especially with Frank likely to have quality options to call upon from the substitutes' bench if required.