Abstract A laboratory bioassay was developed to measure the pathogenicities of isolates of mitosporic fungi to Varroa destructor, an ectoparasite of the European honeybee, Apis mellifera. Forty isolates of entomopathogenic fungi were assessed against V. destructor in a single-dose experiment (conidial concentration 1×108 ml−1) at 25 °C and 100% RH. The fungal species were Verticillium lecanii (nine isolates), Hirsutella spp. (16 isolates), Paecilomyces spp. (three isolates), Beauveria bassiana (four isolates), Metarhizium spp. (six isolates), and Tolypocladium spp. (two isolates). All isolates could infect and kill V. destructor and 26 caused mean times to death of less than 100 h. Control (Tween-treated) mortality was 5% at 7 days post-treatment. Nineteen isolates were also examined for side effects against bees. Caged bees sprayed with conidial suspensions (1×108 ml−1) of seven of these isolates died within 14 days. However, not all mortality could be attributed to fungal infection as confirmed by sporulation; the mortality of control bees was 27%. Nine isolates were selected for further examination against V. destructor at 30 °C and 40% RH to simulate the conditions in bee colonies. Of these, three isolates of M. anisopliae, one of V. lecanii, and one of B. bassiana killed 100% of V. destructor within 7 days at a conidial concentration of 1×108 ml−1. One isolate of M. anisopliae also killed 97% of V. destructor within 7 days at a conidial concentration of 1×106 ml−1. These results indicate that entomopathogenic fungi have potential as microbial control agents of V. destructor in honeybee colonies.