AbstractPassive acoustic monitoring (PAM) is increasingly popular in ecological research, but recording and analyzing large amounts of data is still a critical bottleneck for the long‐term monitoring of multiple species. We evaluated how temporal and spatial sampling effort affects species diversity estimates using a set of 14,045 1‐min recordings from various neotropical birds and anuran communities. Our goals were to evaluate (i) the daily vocal activity cycle of birds and anurans, (ii) the effect of temporal structure (e.g., number of minutes listened each hour; continuous versus intermittent recordings) on determining the species composition, and (iii) the species–area relationship, and how the number of recorders affects species richness estimates. Based on sampling coverage and completeness, we (iv) evaluate manual inspection schedules for birds and anurans across four biomes of Brazil. We found marked diel variation in vocal activity between taxonomic groups, indicating that birds and anurans are more efficiently detected during early periods of the day and night, respectively. For proper diversity estimates, biomes with higher biodiversity required longer inspecting periods and a larger number of replicates, irrespective of taxa. Although fewer recordings per hour are less informative than full‐hour sampling, species diversity is better estimated when inspected minutes are interspersed over longer periods than inspecting minutes recorded over shorter timespans. Based on our findings, we recommend how to set PAM programs over highly diverse ecosystems.Abstract in Portuguese is available with online material.