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Although most predators are generalists, the majority of studies on the association between prey availability and prey consumption have focused on specialist predators. To investigate the role of highly generalist predators in a complex food web, we measured the relationships between prey consumption and prey availability in two common arthropodivorous bats. Specifically, we used high-throughput amplicon sequencing coupled with a known mock community to characterize seasonal changes in little brown and big brown bat diets. We then linked spatiotemporal variation in prey consumption with quantitative prey availability estimated from intensive prey community sampling. We found that although quantitative prey availability fluctuated substantially over space and time, the most commonly consumed prey items were consistently detected in bat diets independently of their respective abundance. Positive relationships between prey abundance and probability of consumption were found only among prey groups that were less frequently detected in bat diets. While the probability of prey consumption was largely unrelated to abundance, the community structure of prey detected in bat diets was influenced by the local or regional abundance of prey. Observed patterns suggest that while little brown and big brown bats maintain preferences for particular prey independently of quantitative prey availability, total dietary composition may reflect some degree of opportunistic foraging. Overall, our findings suggest that generalist predators can display strong prey preferences that persist despite quantitative changes in prey availability., (© 2020 John Wiley & Sons Ltd.)