Most Vibrio species in autochthonous marine microbial communities, such as Vibrio alginolyticus, Vibrio harveyi, Vibrio anguillarum among others, are considered nonpathogenic for humans. However, because many bacterial virulence genes are located in mobile genetic elements, the acquisition of mobile DNA could mediate the appearance of virulent or more virulent strains even in a species defined as nonpathogenic. In this study, we screened a collection of marine nonpathogenic Vibrio strains isolated in the area of the Venetian Lagoon for the presence of virulence and fitness genes usually present in Vibrio cholerae and Vibrio parahaemolyticus clinical isolates. More than one-third of the strains tested positive for the presence of at least one of the potential virulence/fitness genes with the gene encoding the V. cholerae neuraminidase the most frequently detected. Moreover, 13 of the environmental strains carried modified versions of the V. cholerae pathogenicity island VPI-2, and four of them also contained partial fragments of the V. parahaemolyticus Vp-PAI. The data obtained support the view of nonpathogenic Vibrio strains as a significant reservoir of virulence and fitness genes. The emergence of environmental bacteria with new virulence traits might constitute a direct concern for public health and a risk for human health.