Entomopathogenic nematodes (EPNs) are increasingly promoted as sustainable alternatives to synthetic plant protection products, yet their effects on non-target organisms remain poorly characterized. This gap is particularly relevant for soil-dwelling bees that nest and hibernate in soil, where EPNs are commonly applied. Here we show that two commercially used EPN species, Steinernema carpocapsae and Heterorhabditis bacteriophora, can infest and markedly reduce survival of hibernating buff-tailed bumble bee (Bombus terrestris) gynes. Freshly mated queens were exposed to field-realistic EPN concentrations (0.25–1 million infective juveniles m⁻²) under simulated hibernation conditions (14 °C, 60% relative humidity) for 28 days. Both EPN species significantly reduced gyne survival relative to controls (95%), with survival declining to 40–60% for S. carpocapsae and 55–80% for H. bacteriophora (log-rank test, P < 0.001). Survival was independent of S. carpocapsae concentration (P > 0.4), whereas mortality from H. bacteriophora increased significantly with dose (P < 0.01). Both nematode species reproduced successfully within bee cadavers, indicating that hibernating gynes can serve as alternative hosts, although reproduction did not differ among species or concentrations. These findings demonstrate that hibernating bumble bee queens are susceptible to EPN exposure and reveal an overlooked risk to pollinators, particularly given the seasonal overlap between EPN application and queen hibernation. Our results underscore the need for systematic non-target risk assessment of biological control agents.