Passive acoustic methods are increasingly used in biodiversity research and monitoring programs because they are cost-effective and permit the collection of large datasets. However, the accuracy of the results depends on the bioacoustic characteristics of the focal taxa and their habitat use. In particular, this applies to bats which exhibit distinct activity patterns in three-dimensionally structured habitats such as forests. We assessed the performance of 21 acoustic sampling schemes with three temporal sampling patterns and seven sampling designs. Acoustic sampling was performed in 32 forest plots, each containing three microhabitats: forest ground, canopy, and forest gap. We compared bat activity, species richness, and sampling effort using species accumulation curves fitted with the clench equation. In addition, we estimated the sampling costs to undertake the best sampling schemes. We recorded a total of 145,433 echolocation call sequences of 16 bat species. Our results indicated that to generate the best outcome, it was necessary to sample all three microhabitats of a given forest location simultaneously throughout the entire night. Sampling only the forest gaps and the forest ground simultaneously was the second best choice and proved to be a viable alternative when the number of available detectors is limited. When assessing bat species richness at the 1-km 2 scale, the implementation of these sampling schemes at three to four forest locations yielded highest labor cost-benefit ratios but increasing equipment costs. Our study illustrates that multiple passive acoustic sampling schemes require testing based on the target taxa and habitat complexity and should be performed with reference to costbenefit ratios. Choosing a standardized and replicated sampling scheme is particularly important to optimize the level of precision in inventories, especially when rare or elusive species are expected.