The two teams will be locking horns for the first time since July 2018 when the U's secured a 2-1 friendly victory against the Lions.
Match preview
Millwall will be competing in the second tier of English football for the sixth successive season after narrowly missing out on a Championship playoff spot last season.
The Lions were in the hunt to secure a top-six finish at the back end of the campaign, but they eventually finished ninth in the table - two places higher than the previous year - and six points behind Luton Town in sixth.
Gary Rowett's side managed to get some of their transfer business done early in the window, with club-record signing Zian Flemming the most notable addition from Fortuna Sittard, while George Honeyman, Benik Afobe and Charlie Cresswell (on loan) have also joined the club.
The loss of star winger Jed Wallace - who has joined West Bromwich Albion on a free transfer - will be felt, however, while both Mahlon Romeo and Connor Mahoney have also left The Den.
Millwall began their pre-season with an 5-0 win against Bromley, and they were then on the wrong end of an eventful 5-4 defeat at Crystal Palace, before returning to winning ways with a 2-0 victory over Dartford on Tuesday.
After Friday's clash with Colchester, the Lions are scheduled to face Watford, Hammarby IF and Ipswich Town before beginning the 2022-23 Championship season at home against Stoke City on July 30.
Colchester United will be looking to improve on last season's 15th-place finish in League Two as they prepare for a seventh-successive season in the fourth tier.
The U's ended the campaign strongly with five wins and one draw in their final eight league games, and head coach Wayne Brown is now hoping to build another good run of form in pre-season ahead of their opening League Two fixture against Northampton Town on July 30.
Colchester are yet to make waves in the transfer market this summer as they have only recruited winger Alex Newby from Rochdale and goalkeeper Sam Hornby from Bradford City, and they may need to strengthen their first-team squad further if they wish to mount a playoff push this season.
Brown's men began their pre-season with contrasting results against Swansea City, winning their first match 2-0 before suffering a slender 1-0 defeat. Since then, the U's followed up a 2-0 loss away at Reading with a comfortable 3-1 victory over Bath City and a 4-2 defeat against Charlton Athletic on Tuesday.
Colchester, who have won only two of their last seven meetings with Millwall, will be keen to return to winning ways on Friday before facing Southend United and Ebbsfleet United in their final two warm-up fixtures.
Millwall Club Friendlies form:
Colchester United Club Friendlies form:
Team News
Millwall boss Rowett has named completely different starting lineups in his last two friendlies and he is expected to make several changes again on Friday.
Afobe, who has scored three goals in his last two warm-up matches, is set to be involved again, as will fellow summer recruits Cresswell and Flemming who have also chipped in with goals in pre-season.
As for Colchester, Brown handed 11 of his players 60 minutes of game time against Charlton before making 11 substitutions for the final half an hour, and a similar approach could be taken on Friday.
The likes of Hornby, Noah Chilvers, Junior Tchamadeu and John Akinde, who all began on the bench last time out, could be handed starts on this occasion.
Millwall possible starting lineup:
Bialkowski; Cresswell, Wallace, Cooper; McNamara, Saville, Mitchell, Malone; Honeyman, Flemming; Afobe
Colchester United possible starting lineup:
Hornby; Tchamadeu, Chambers, Smith, Clampin; Newby, Skuse, Huws, Judge; Sears, Akinde
We say: Millwall 2-0 Colchester United
While it remains to be seen how both sides will line up on Friday, Millwall will be regarded as the favourites to come out on top.