Anopheles barberi is most often found associated with woodlands. The adults usually emerge in June and can be found resting during the day under bridges, in culverts, and in buildings in or near wooded areas. The female will readily enter houses and is a persistent but nervous biter, rarely finishing a blood meal in a single sitting. The larvae develop in rot cavities of trees, stumpholes, and occasionally in artificial containers in or near wooded areas. The species overwinters in the larval stage and may be found frozen in ice within treeholes.