Richie Wellens's side succumbed to the superiority of Arsenal's Under-21s in their tournament opener, while the visitors got the better of MK Dons by two goals to one.
Match preview
In front of a 2,100-strong crowd on September 3, Leyton Orient came out second best against the current crop of Arsenal's Hale End talents, falling to a 2-1 loss despite Dan Happe's first-half strike.
The Orient defender's equaliser came after Josh Robinson broke the deadlock for the Gunners with just three minutes on the board, but Ismeal Kabia restored Arsenal's lead early in the second period to condemn the League One outfit to the worst possible start.
Defeat to Arsenal did actually prove to be the catalyst for a change in League One fortunes for Leyton Orient, who went unbeaten in four straight league matches after being downed by the Gunners, albeit while also losing to Brentford in the EFL Cup.
However, Wellens's men have experienced a dampening start to October with back-to-back defeats against Exeter City (1-0) and Lincoln City (2-1), the latter of which saw Tuesday's hosts fail to make their first-half dominance pay before being outplayed in the second.
Winless in four successive League One games, Leyton Orient are hovering dangerously close to the third-tier relegation line in 19th place, and not since August's 4-1 EFL Cup thrashing of Newport County have they actually won a game at home, failing to prevail in five on the spin since then.
On the other side of the coin, Colchester United were on the correct end of a 2-1 scoreline in their first EFL Trophy showdown of the season, despite finding themselves a goal behind against MK Dons when Stephen Wearne found the mark.
Responses from Anthony Scully and Fiacre Kelleher ensured that the League Two outfit would be sitting pretty in the top two of the standings after one matchday; their current second-placed ranking behind Arsenal would send them straight into the second round.
Inconsistency has plagued Danny Cowley's men since that opening EFL Trophy success, though, as while they immediately followed that 2-1 win up with a 3-0 trouncing of Tranmere Rovers, they are now winless in three straight fourth-tier matches.
A 4-0 humbling at Walsall on September 28 preceded a pair of draws with Port Vale (1-1) and Carlisle United (0-0), and the 16th-placed League Two side are still pursuing their first away win of the season, as well as their first clean sheet on the road since January.
Familiar foes over the past couple of decades, Leyton Orient and Colchester last did battle in the 2022-23 League Two campaign, where the O's took four points from a possible six with a 3-1 away win before a 2-2 stalemate at Tuesday's venue.
Leyton Orient EFL Trophy form:
L
Leyton Orient form (all competitions):
L
W
D
D
L
L
Colchester United EFL Trophy form:
W
Colchester United form (all competitions):
D
W
W
L
D
D
Team News
Leyton Orient have only lost two players to October international call-ups, as young goalkeeper Josh Keeley - on loan from Tottenham Hotspur - has been called up to the Republic of Ireland Under-21 team, while Ethan Galbraith will represent Northern Ireland.
Middlesbrough-owned Zach Hemming should therefore start in between the posts on Tuesday, as Sam Howes - who started the loss to Arsenal's Under-21s - was loaned to Wealdstone last month.
Midfielder Sean Clare and defender Jack Simpson both failed to make it to the hour mark at the weekend; Jordan Brown could now come into contention to replace the former.
Colchester have also lost a couple of troops to national team duty, as striker Lyle Taylor has been called up to the Montserrat squad, while Aaron Donnelly and Jamie McDonnell will link up at the Northern Ireland Under-21 camp.
Portsmouth loanee Scully could start in place of Taylor in the final third, but another summer arrival from Fratton Park - ex-Arsenal goalkeeper Matt Macey - may drop out for Tom Smith.
Owura Edwards (hamstring), Teddy Bishop (ankle) and Harry Anderson (groin) all trained before the draw with Carlisle, but only the latter made the matchday squad and was an unused substitute anyway.
Leyton Orient possible starting lineup:
Hemming; James, Beckles, Simpson, Currie; Pratley; O'Neill, Brown, Perkins, Agyei; Donley
Colchester United possible starting lineup:
Smith; Hunt, Kelleher, Flanagan, Iandolo; Payne, Read, Bishop, Gordon; Scully, Tovide
We say: Leyton Orient 1-0 Colchester United
Home may not be where the heart is for Leyton Orient right now, but Wellens's men are squaring up to a Colchester side who have gone almost nine months without a competitive clean sheet away from home.