Mobile hydroacoustic surveys are routinely used to estimate pelagic fish abundance. In the Great Lakes, alewife are commonly surveyed with mobile hydroacoustics, however, their behavior often has them associated with epilimnetic habitats which increases the potential for vessel avoidance to bias hydroacoustic observations. Abundance estimates from mobile hydroacoustic surveys are typically made using depth and size thresholds to classify targets to species. In hydroacoustic surveys, fish can appear to be smaller when oriented off horizontal axis, as is common with fish displaying vessel avoidance behaviour. This presents a problem where alewives that are diving may appear too small to be correctly classified. We compared alewife swimming behaviours and target strength distributions between stationary up-looking and mobile down-looking hydroacoustics to quantify how vessel avoidance may bias hydroacoustic observations. Alewives from the mobile survey were found to be diving away from the surface, moving faster and in more linear paths than the undisturbed fish from the stationary survey. In the mobile survey, alewives were observed at smaller target strength values than would be expected if boat avoidance was not occurring. Our results suggest that alewife behavioral changes associated with vessel avoidance affect the number and size of targets classified as alewife from mobile hydroacoustic surveys in Lake Ontario.