Bacterium is still a major cause of many infectious diseases and a global threat to human health, aquaculture, and animal feeding. Prevention by vaccination is the most efficient and economical way of fighting bacterial diseases, but one of the persistent challenges to prevent bacterial infections and disease transmissions is the existence of multiple bacterial species, families, and genera and the lack of efficient polyvalent vaccines against them. The information on candidate immunogens for polyvalent vaccine development is elusive, as well. For the development of broad cross-protective vaccines, we have employed heterogeneous antiserum-based immunoproteomics approaches to identify antigenically similar outer membrane (OM) proteins that could be used as potential polyvalent vaccine candidates against Vibrio parahaemolyticus , V. alginolyticus , V. fluvialis , Aeromonas hydrophila , and A. sobria infections. VPA1435, VP0764, VPA1186, VP1061, and VP2850 could be recognized by at least three antisera and demonstrated significantly passive and active immune protection against V. parahaemolyticus infection in a crucian carp model. VP1061 and VP2850 induced higher immune and protective abilities than the other three OM proteins. Furthermore, the abilities of VP1061 and VP2850 in the generation of broad cross-protective immune reaction against the infections of V. alginolyticus , A. hydrophila , and Pseudomonas fluorescens were also investigated in fish and mouse models. Our results suggested that VP1061 and VP2850 could potentially be used as polyvalent vaccine candidates for the development of novel polyvalent vaccines against V. parahaemolyticus and other Gram-negative pathogens. On the basis of these results, characteristics of OM proteins as polyvalent vaccine candidates have been addressed.