These data include in situ images of lake trout (Salvelinus namaycush) and round goby (Neogobius melanostomus) collected by an autonomous underwater vehicle (AUV) in Lakes Michigan and Superior in 2020 and 2021 and laboratory images of round goby collected by a GoPro in 2021. These images were collected and labeled to train and validate an algorithm for automated fish sizing and biomass estimation. The dataset includes original images, ground truth binary masks, where the designated benthic fish is represented by white pixels and the background by black pixels, and calibration images to correct any type of distortion on the imagery if needed.