Faunal mortality from roadkill has a negative impact on global biodiversity, and bats are among the roadkilled animals. In South America, the Atlantic Forest covers southeastern Brazil, a region which sustains a large bat diversity. In this biome, the Sooretama reserves are crossed by the federal highway BR-101, one of the busiest in Brazil. We analyzed bats roadkilled along the 25 km stretch of highway that crosses the Sooretama reserves. Data were collected between the years 2010 and 2015. In total, 773 individuals distributed among 47 bat species were roadkilled during this period. The insectivorous feeding guild was the most affected, accounting for 25 species and 74% of the recorded roadkill, and those flying in the open area were the most frequently roadkilled (41.9%). Bat mortality rates did not differ between months of the year. However, the relation between rainy days and roadkill was negative. Monitoring by foot was more efficient than by car for detection of bat carcasses. Radars with a speed limit below 60 km/h reduced the rates of roadkill. The diversity of deceased bats found in this study represents 40% of the known species in the Atlantic Forest, and is the largest among current studies of species killed on highways globally. The present study raises concerns about the high diversity and abundance of roadkilled insectivorous bats and the conservation of these animals in the Neotropical region.