In this study, a total of 54 Vibrio alginolyticus strains were analyzed. The isolates were recovered from different compartments of the Ruditapes decussatus hatchery sized in the National Institute of Marine Sciences and Technologies, Monastir, Tunisia. All isolates were biochemically identified (API 20E and API ZYM strips), characterized by amplification of the Hsp-40 gene polymerase chain reaction (PCR) and analyzed by enterobacterial repetitive intergenic consensus (ERIC)-based genotyping to evaluate genetic relationship between the isolated strains. We also looked for the presence of ten V. cholera virulence genes (toxRS, toxR, toxT,toxS, tcpP, tcpA, ace, vpi, zot and ctxA) in the genomes of Vibrio isolates. The antibiotics susceptibility, exoenzymes production and in vitro cytotoxic activities against HeLa cell line were also carried out for all tested bacteria. Most of V. alginolyticus isolates showed noteworthy antimicrobial resistance rates to at least ten antibacterial agents. For most isolates, the minimum inhibitory concentration (MIC) data showed that tetracyclin and streptomycin were the most effective antibiotics. Construction of the phylogenetic dendogram showed that studied isolates were in general genetically heterogeneous; however some Vibrio strains were present in different structures of the R. decussatus hatchery. The V. cholerae virulence genes investigation showed a wild distribution of toxS (49/54), toxR (45/54) and toxT (22/54) genes among V. alginolyticus strains isolated from the R. decussatus rearing system. Cytotoxic effects of several Vibrio extracellular products (28/54) were also observed on HeLa cells.