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Due to increasing threats facing bats, long-term monitoring protocols are needed to inform conservation strategies. Effective monitoring should be easily repeatable while capturing spatio-temporal variation. Mobile acoustic driving transect surveys (‘mobile transects’) have been touted as a robust, cost-effective method to monitor bats; however, it is not clear how well mobile transects represent dynamic bat communities, especially when used as the sole survey approach. To assist biologists who must select a single survey method due to resource limitations, we assessed the effectiveness of three acoustic survey methods at detecting species richness in a vast protected area (Everglades National Park): (1) mobile transects, (2) stationary surveys that were strategically located by sources of open water and (3) stationary surveys that were replicated spatially across the landscape. We found that mobile transects underrepresented bat species richness compared to stationary surveys across all major vegetation communities and in two distinct seasons (dry/cool and wet/warm). Most critically, mobile transects failed to detect three rare bat species, one of which is federally endangered. Spatially replicated stationary surveys did not estimate higher species richness than strategically located stationary surveys, but increased the rate at which species were detected in one vegetation community. The survey strategy that detected maximum species richness and the highest mean nightly species richness with minimal effort was a strategically located stationary detector in each of two major vegetation communities during the wet/warm season.