Relatively little is known about bats or bat hibernacula in northern Canada. We were interested in documenting species diversity and seasonal activity of bats in Wood Buffalo National Park, including use of a cave hibernaculum by Little Brown Myotis (Myotis lucifugus), Northern Myotis (Myotis septentrionalis), and Big Brown Bats (Eptesicus fuscus). We used acoustic monitoring and mist netting over 3 y to assess species diversity and seasonal activity. We also recorded cave temperature during hibernation. During the summers of 2010 to 2012, we captured 470 bats including M. lucifugus, M. septentrionalis, and E. fuscus. We identified 2 migratory species via echolocation recordings in 2011: Lasiurus cinereus (Hoary Bat) was recorded in the area from mid-May to early October, and Lasiurus borealis (Eastern Red Bat) from mid-June to early August. Resident bat activity at the hibernaculum was greatest from mid-June to early September. Our findings provide a first approximation of species diversity and describe seasonal activity patterns of bats in Wood Buffalo National Park.