Acoustic deterrents have shown potential as a viable mitigation measure to reduce human impacts on bats; however, the mechanisms underpinning acoustic deterrence of bats have yet to be explored. Bats avoid ambient ultrasound in their environment and alter their echolocation calls in response to masking noise. Using stereo thermal videogrammetry and acoustic methods, we tested predictions that: (i) bats would avoid acoustic deterrents and forage and social call less in a ‘treated airspace’; (ii) deterrents would cause bats to fly with more direct flight paths akin to commuting behaviour and in line with a reduction in foraging activity, resulting in increased flight speed and decreased flight tortuosity; and (iii) bats would alter their echolocation call structure in response to the masking deterrent sound. As predicted, overall bat activity was reduced by 30% and we recorded a significant reduction in counts of Pipistrellus pygmaeus (27%), Myotis spp. (probably M. daubentonii) (26%), and Nyctalus spp. and Eptesicus spp. (68%) passes. Pipistrellus pygmaeus feeding buzzes were also reduced by the deterrent in relation to general activity (by 38%); however, social calls were not (only 23% reduction). Bats also increased their flight speed and reduced the tortuosity of their flight paths, and P. pygmaeus reduced echolocation call bandwidth and start frequency of calls in response to deterrent playback, probably owing to the masking effect of the sound. Deterrence could therefore be used to remove bats from areas where they forage, for example wind turbines and roads, where they may be under threat from direct mortality.