Chiellini missed the Serie A leaders' game against Udinese on Saturday because of a thigh injury and Juve said his condition was "being monitored" after he was left out of the squad for the match in France.
Mandzukic was also omitted because of a "muscular strain in his right adductor region", the club said on Monday.
Midfielder Claudio Marchisio also misses out as he continues his rehabilitation after picking up a knee injury in April.
Juve lead Group H on four points with Lyon in third place one point behind.
Despite the injury setbacks, the visitors are confident they can grab a good result in Lyon.
"At Juve, every game is vital. We are confident we can live up to expectations," midfielder Miralem Pjanic, who played at OL from 2008-11, told a news conference.
"We're missing some key players but every good team should be able to roll with the punches and adapt," said coach Massimiliano Allegri.
Lyon, who lost 2-0 at Ligue 1 leaders Nice at the weekend, look set to start up front with France internationals Nabil Fekir and Alexandre Lacazette, who is back from injury.
"Tomorrow's game will not just be about the Lazazette-Fekir pair. We will need to be rigorous," coach Bruno Genesio said.
"Defence is key to win games on the long term. We need to stick together."
(Writing by Julien Pretot; Editing by Ed Osmond)
