Bats have deserved much scientific attention due to their biological-ecological properties and increasingly recognized epidemiological significance. Ecdysteroids are insect-molting hormones that (under experimental conditions) have stimulating and anabolic effects in mammals, including humans. Therefore, these biologically active compounds are currently under consideration by the World Anti-Doping Agency to become doping-controlled substances. Previously we demonstrated that low to high concentrations of ecdysteroids appear in the blood of insectivorous passerine birds. Since passerine birds and echolocating bats share several adaptive mechanisms in connection with flying, and insectivorism is also among their common traits, we hypothesized that ecdysteroids might also be present in the blood of insectivorous bats. To test this hypothesis, blood samples of eight insectivorous bat species were collected and analyzed for the presence of ecdysteroids with highly sensitive targeted ultra-high-performance liquid chromatography coupled to high-resolution quadrupole-orbitrap mass spectrometry method (UHPLC-HRMS). The results supported our hypothesis, because nine ecdysteroids were detected in bat blood. The spectrum of these ecdysteroids was similar in those bat species which have their most preferred food items from the same insect order, supporting insects as the most likely source of these hormones. It was also shown that the spectrum of blood-borne ecdysteroids was broader in the autumn than in the summer, and higher concentrations of 20-hydroxyecdysone were measured in samples of large size bat species in comparison with small size ones. Based on the known physiologic effects of ecdysteroids, we postulate that these results might have implications on the metabolic rate and parasite burdens of insectivorous bats.