Understanding population trends of any species is essential for conservation and management. However, due to difficulty in sampling some species, population status of many bat species is poorly understood. In an effort to resolve this issue, especially in light of emerging threats (e.g., white-nose syndrome and wind energy), a national mobile acoustic monitoring protocol, modeled after European programs, was developed to survey summer bat populations in the United States. While the program calls for conducting transects along roadways, some have suggested that waterways may allow for the gathering of more information. Therefore, we quantified species richness and abundance along car and boat transects to identify the most efficient mobile method. Furthermore, to compare the capabilities of mobile acoustic transects to a more traditional and better understood survey method, we compared species density along transects to stationary acoustic detectors. Using sample-based rarefaction, there was no difference at the 95% confidence level in species density (species/sample) between methods, however stationary points accumulated species more quickly than mobile methods. Of the mobile transect methods, car transects had higher diversity indices than boat transects and tended to show slightly higher species density. While over 1.5 times as many calls were recorded and identified along boat transects, there were no clear advantages of boat transects for monitoring bats except for Myotis grisescens. Additionally, car transects were least time consuming, leading us to conclude that car transects are the most efficient mobile acoustic method to monitor species. Mobile acoustic transects can likely monitor 2–4 species in the Eastern United States, including species with no current population monitoring methodology.