Ballast water is a significant vector of microbial dissemination; however, biofouling on commercial vessel hulls has been poorly studied with regard to pathogenic bacteria transport. Biofouling on three commercial vessels and seven port structures in Ensenada, Baja California, Mexico, was examined by qPCR to identify and quantify Vibrio parahaemolyticus, a worldwide recognized food-borne human pathogen. Pathogenic variants (trh+, tdh+) of V. parahaemolyticus were detected in biofouling homogenates samples from several docks in Ensenada and on the hulls of ships with Japanese and South Korean homeports, but not in reference sampling stations. A total of 26 tdh+ V. parahaemolyticus colonies and 1 ORF8+/O3:K6 strain were also isolated from enriched biofouling homogenate samples confirming the qPCR analysis. Our results suggest that biofouling is an important reservoir of pathogenic vibrios. Thus, ship biofouling might be an overlooked vector with regard to the dissemination of pathogens, primarily pathogenic V. parahaemolyticus.