Although hydroacoustic technologies are widely used in fisheries research, there are few studies examining the effects of Marine Protected Areas (MPAs) using hydroacoustic methods. We help close this knowledge gap by using hydroacoustics to examine the effects of habitat type and closure to fishing, on fish populations in the Cabo Pulmo National Park (CPNP). Fish density (fish/ha) was significantly higher for transects within the CPNP compared to those outside in non-MPA control areas (447 ± 141 S.E.M vs 112 ± 19, respectively), and higher still over reef-specific transects inside the park (5388 ± 1282). Largest mean fish size was also found over reef-specific transects, followed by mixed habitats inside the park, then the rocky and finally sandy control sites outside the park. Acoustic fish biomass estimates differed significantly between sites ranging from 1.88 t/ha over the CPNP reef-specific transects to 0.01 t/ha at the sandy control site. Acoustic estimates of fish biomass did not differ significantly from those calculated using Underwater Visual Census (UVC) data, although densities did, due to higher fish numbers in the smallest UVC size classes. This study is a valuable starting point in demonstrating the utility of hydroacoustics for the assessment of fish in coastal MPAs.