Hernan Torres's side progressed courtesy of a second-placed finish in Group D, while their unbeaten visitors cruised through as Group H winners.
Match preview
By reaching the last-16 stage of the tournament, Deportes Tolima have already ensured that the 2022 Copa Libertadores will represent their best-ever showing on the continental stage.
The recently-dethroned Colombian champions fended off competition from America Mineiro and Independiente del Valle to finish second in Group D behind Atletico Mineiro, who only progressed in first place courtesy of a superior goal difference.
Torres's men would quickly recover from an opening-day loss to the group winners to go unbeaten in their next five Group D matches, and they sealed their place in the knockout rounds with a 2-1 victory over Atletico Mineiro on the final matchday.
The hosts also head into the first leg of their last-16 tie having lost just two of their last 18 matches in all tournaments, although one of those came against Atletico Nacional earlier this week, as their Colombian top-flight title was ripped from their grasp over a two-legged tie.
In contrast, Flamengo's continental nous at this level is well-documented, as the Brazilian giants seek to win South America's top honour for the third time and end a three-year drought in the competition.
Dorival Junior's side were one of only three teams to make it through to the last-16 stage without losing a game - the other being River Plate and defending champions Palmeiras - and they only dropped two points en route to top spot in Group H.
Flamengo's domestic fortunes did take an alarming nosedive following the conclusion of the group stage, though, as they suffered five defeats in six Brasileiro and Copa do Brasil matches before bouncing back with a 3-0 success over America Mineiro on Saturday.
Junior's side made it through to the last-16 by scoring at least two goals in all of their group-stage matches, but they have bowed out at this point of the Copa Libertadores in the last two campaigns in which they have failed to make the final.
Deportes Tolima Copa Libertadores form:
Deportes Tolima form (all competitions):
Flamengo Copa Libertadores form:
Flamengo form (all competitions):
Team News
Deportes Tolima saw attacker Andres Ibarguen leave the field after just 18 minutes against Atletico Nacional with an apparent leg injury, which is expected to rule him out of the first leg.
Juan David Rios and Anderson Angulo also occupy spots in the hosts' treatment room, while creative hub Daniel Catano will be fine to take part despite seeing red in league action at the weekend.
Luis Miranda will likely fill in for Ibarguen on the right flank for Wednesday's game, as Michael Rangel and Juan Caicedo battle to start at the tip of the attack.
Meanwhile, Flamengo were forced to take off Joao Gomes in the dying embers of Saturday's success over America Mineiro, and he will also miss the weekend's showdown with Santos through suspension.
Former Arsenal and Chelsea centre-back David Luiz is still struggling with a thigh problem alongside Bruno Henrique, whose knee injury could still keep him out for a number of weeks.
Junior may be loath to disrupt the harmony too much after his side managed to snap their losing streak in emphatic fashion last time out, and Manchester United loanee Andreas Pereira could be set for one of his final games for the club before his return to Old Trafford.
Deportes Tolima possible starting lineup:
Dominguez; Marulanda, Quinones, Moya, Hernandez; Trujillo, Urena; Miranda, Catano, Lucimi; Rangel
Flamengo possible starting lineup:
Santos; Rodinei, Pablo, L. Pereira, Henrique; A. Pereira, Arao, Maia; De Arrascaeta; Barbosa; Pedro
We say: Deportes Tolima 0-3 Flamengo
Deportes Tolima's progress to the last-16 stage ought to be celebrated as the success it is for the Colombian outfit, but the quality of Flamengo should shine through here.