AbstractDuring the last decades, the use of bioacoustics as a non‐invasive and cost‐effective sampling method has greatly increased worldwide. For bats, acoustic surveys have long been known to complement traditional mist‐netting, however, appropriate protocol guidelines are still lacking for tropical regions. Establishing the minimum sampling effort needed to detect ecological changes in bat assemblages (e.g., activity, composition, and richness) is crucial in view of workload and project cost constraints, and because detecting such changes must be reliable enough to support effective conservation management. Using one of the most comprehensive tropical bat acoustic data sets, collected in the Amazon, we assessed the minimum survey effort required to accurately assess the completeness of assemblage inventories and habitat selection in fragmented forest landscapes for aerial insectivorous bats. We evaluated a combination of 20 different temporal sampling schemes, which differed regarding number of hours per night, number of nights per site, and sampling only during the wet or dry season, or both. This was assessed under two different landscape scenarios: in primary forest fragments embedded in a matrix of secondary forest and in the same forest fragments, but after they had been re‐isolated through clearing of the secondary forest. We found that the sampling effort required to achieve 90% inventory completeness varied considerably depending on the research aim and the landscape scenario evaluated, averaging ~80 and 10 nights before and after fragment re‐isolation, respectively. Recording for more than 4 h per night did not result in a substantial reduction in the required number of sampling nights. Regarding the effects of habitat selection, except for assemblage composition, bat responses in terms of richness, diversity, and activity were similar across all sampling schemes after fragment re‐isolation. However, before re‐isolation, a minimum of four to six sampling hours per night after dusk and three to five nights of sampling per site were needed to detect significant effects that could otherwise go unnoticed. Based on our results, we propose guidelines that will aid to optimize sampling protocols for bat acoustic surveys in the Neotropics.