Distribution and abundance of two temperate-zone insectivorous bats, Daubenton's (Myotis daubentonii) and common pipistrelle (Pipistrellus pipistrellus), and their potential prey were studied along an altitudinal river gradient in relation to environmental variables including air temperature, wind speed, water surface state, and presence or absence of bank-side trees. Using a Latin square design at ten different habitat combination types, ultrasound recordings and insect sampling were carried out to quantify bat habitat preferences and potential prey abundance and classification. Myotis daubentonii and P. pipistrellus activity was significantly higher over smooth water river sections with trees on either or both banks while cluttered and rapid water sections were avoided. Conversely, insect abundance was not related to water surface condition or the presence or absence of bank-side trees. Nematoceran dipterans made up 98% of insect numbers, with small numbers of brachycerans and cyclorrhaphans. The most common insect families were Chironomidae and Ceratopogonidae. There was no correlation between bat activity and aerial insect activity, suggesting that aerial prey availability is not the sole driver of bat habitat choice. Bat and insect abundance were each correlated positively with night-time air temperature. No bat passes or flying insects were recorded at temperatures < 4°C. At 5°C, only M. daubentonii were observed foraging, and at 6°C there were more M. daubentonii present than any other bat species. No correlation was found between number of bat passes hr-1 and wind speed, moon visibility, moon phase, and percentage cloud cover. Rain did not affect M. daubentonii, but P. pipistrellus preferred to forage on dry nights. Bats were predicted to forage preferentially where aerial insect abundance was highest but this was found to not be case, and other aspects such as detection of prey against clutter may have an important role to play in habitat choice.