Shrimp production has been affected by disease outbreaks caused by Vibrio sp. bacteria. Using a polyculture system has been proposed as preventive management, but until now, the mode of action by which the organisms in polyculture obtain some benefits was unknown. Some studies indicate that these benefits are related to the immune system in the skin mucus. The present study aimed to determine the antibacterial potential of skin mucus in two tilapia varieties, Oreochromis sp. (marine adapted) and O. niloticus (freshwater), against Vibrio bacteria that affect the shrimp Penaeus vannamei. Skin mucus extracts were obtained from both varieties of tilapia during spring and winter. The extracts were: acidic, aqueous and two organics. In total, 16 extracts were obtained. During the winter season, no antibacterial activity was detected; however, in the spring, the acidic extract from the marine-adapted tilapia inhibited V. parahaemolyticus. Additionally, V. harveyi was inhibited by the acidic and organic extracts from both varieties. The aim of this study was confirmed: tilapia skin mucus has antibacterial activity against Vibrio bacteria, which depends on the tilapia variety, bacterial strain, season and the concentration of colony forming units. SDS-PAGE revealed a protease of 96 kD present in all extracts, even in those without antibacterial activity.