Fiorentina coach Vincenzo Montella spent 10 years at Roma during his playing days and also had a spell as caretaker coach at Stadio Olimpico, but there will be no room for sentiment when his side attempt to dump the Eternal City club out of Europe.
Montella's side, who knocked out Tottenham in the round of 32, will be at home for the first leg on March 12 and travel to Rome a week later.
Fiorentina knocked Roma out of the Coppa Italia earlier this month, so Rudi Garcia's side have an opportunity to exact revenge.
Roma beat Feyenoord among a backdrop of crowd disturbances on Wednesday, winning 2-1 on the night and 3-2 on aggregate.
There will also be an all-Spanish tie in last 16, with holders Sevilla taking on Villarreal.
Sevilla were crowned champions when they beat Benfica last season and Unai Emery's side knocked out Borussia Monchengladbach to keep their hopes of lifting the trophy again alive.
Villarreal, conquerors of Salzburg in the round of 32, will be at home in the first leg.
Draw in full:
Everton v Dynamo Kiev
Dnipro v Ajax
Zenit v Torino
Wolfsburg v Inter
Villarreal v Sevilla
Napoli v Dinamo Moscow
Club Brugge v Besiktas
Fiorentina v Roma
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