Gareth Taylor’s side will play the first leg at home on 3/4 March before travelling to Italy for the reverse fixture on 10/11 March.
The pair have never met before in professional competition.
Due to travel restrictions, the seeded and unseeded teams were split into two groups and therefore only able to draw one of four teams. City could only face either Fiorentina, Atletico Madrid, Brondby or Sparta Prague.
Semi-finalists in 2017 and 2018, City reached the last 16 stage with convincing victories over Göteborg FC in the Round of 32.
Georgia Stanway's 50th goal and a Sam Mewis header on her Champions League debut secured a hard-fought away win, and City cruised to a 3-0 home triumph in the second leg, thanks to a Lauren Hemp wonderstrike and Stanway brace.
Fiorentina will be hoping to reach the quarter-finals for the first time.
The Italian side eliminated Slavia Praha 3-2 on aggregate in the Round of 32, thanks to Daniele Sabatino's late strike.
The tie will see Dutch midfielder Tessel Middag face her former Club.
Fixture information will be confirmed in due course...
Wolfsburg (GER) v LSK Kvinner (NOR)
Barcelona (ESP) v Fortuna Hjorring (DEN)
Rosengard (SWE) v SKN St Polten (AUT)
BIIK-Kazygurt (KAZ) v Bayern Munich (GER)
Manchester City (ENG) v Fiorentina (ITA)
Sparta Prague (CZE) v Paris Saint-Germain (FRA)
Olympique Lyonnais (FRA) v Brondby IF (DEN)
Chelsea (ENG) v Atletico Madrid (ESP)
Round of 16: 3/4 & 10/11 March
Quarter-final & semi-final draw: 12 March
Quarter-finals: 23/24 March & 31 March/1 April
Semi-finals: 24/25 April & 1/2 May
Final: 16 May (Gamla Ullevi, Gothenburg)