In the present study, we investigated the presence of four Vibrio cholerae virulence genes (ctxA, VPI, Zot and ace) in 36 Vibrio alginolyticus isolates obtained from different seawater, sediments and aquatic organisms. We tested the virulence of 13 V. alginolyticus strains against juveniles of Sparus aurata and this virulence was correlated with the presence of V. cholerae virulence genes. A positive amplification for the virulence pathogenicity island was produced by five V. alginolyticus strains and four for cholerae toxin. Some of the V. alginolyticus strains are pathogenic to aquatic animals and might have derived their virulence genes from V. cholerae. V. alginolyticus strains can be considered as a possible reservoir of V. cholerae virulence genes.