Richness is a key variable for understanding biodiversity, and estimates of species occurrence should be as comprehensive as possible and based on reliable records. The employment of diversified methods to be able to detect species, and of adequate sampling efforts is crucial for estimating the richness of bats occurring in highly complex tropical forests. We herein analyzed the richness of bats from one of the largest remnants of the semideciduous Atlantic Forest from Southeastern Brazil, the state conservation park called Parque Estadual do Rio Doce (PERD), based on data from the 1990s and from 2012 to 2016. Based on mixed sampling techniques, including ground-level and canopy nets, acoustic surveys, and roost searching we have revealed that this single state reserve of PERD has the highest bat richness recorded to date among semideciduous Atlantic Forest locations. A total of 54 bat species have been recorded for PERD, including two recently described species from the genera Thyroptera (Thyropteridae) and Eumops (Molossidae). Our data reinforce the value of diversifying sampling methods and the role of PERD as key area for the conservation of the Atlantic Forest biome.