Mobile acoustic surveys are a common method of surveying bat communities. However, there is a paucity of empirical studies exploring different methods for conducting mobile road surveys of bats. During 2013, we conducted acoustic mobile surveys on three routes in north-central Indiana, U.S.A., using (1) a standard road survey, (2) a road survey where the vehicle stopped for 1 min at every half mile of the survey route (called a “start-stop method”), and (3) a road survey with an individual using a bicycle. Linear mixed models with multiple comparison procedures revealed that when all bat passes were analyzed, using a bike to conduct mobile surveys detected significantly more bat passes per unit time compared to other methods. However, incorporating genus-level comparisons revealed no advantage to using a bike over vehicle-based methods. We also found that survey method had a significant effect when analyses were limited to those bat passes that could be identified to genus, with the start–stop method generally detecting more identifiable passes than the standard protocol or bike survey. Additionally, we found that significantly more identifiable bat passes (particularly those of the Eptesicus and Lasiurus genera) were detected in surveys conducted immediately following sunset. As governing agencies, particularly in North America, implement vehicle-based bat monitoring programs, it is important for researchers to understand how variations on protocols influence the inference that can be gained from different monitoring schemes. Vehicle-based mobile acoustic surveys of bats have become a common tool for assessing bat communities and population change across the globe. We assessed the impact that both the timing of these surveys as well as modifications to the standard protocol had on the ability of such surveys to effectively sample a bat community. We found that surveys started shortly after sunset recorded more high-quality bat calls than those begun later in the night and that a mobile protocol that implemented periodic stops was more effective per unit time at recording identifiable bat echolocation calls than the standard method or a bike-based protocol.