A comprehensive study was done to determine the genetic relatedness and the biofilm formation ability of Aeromonas hydrophila strains (n=25) isolated from fish (150 tilapia and 144 mugil) and human stool samples (n=102). The results revealed that Random Amplification of Polymorphic DNA (RAPD-PCR) classified A. hydrophila strains into seven distinct profiles (R1-R7), the amplicon sizes ranged from 183-2930 bp. The isolates were grouped into five main clusters, the presence of isolates from fish and humans in the same cluster indicates the possibility of cross contamination. Biofilm results showed that 96% of A. hydrophila isolates were biofilm producers. At 35ºC, 16 (64%) and 8 (32%) showed strong and moderate biofilm production ability, respectively. At 25ºC, 21 (84%) were biofilm producers, of which, 8 (32%), 7 (28%) and 6 (24%) were strong, moderate and weak, respectively. At 4ºC, decreased biofilm production ability was noticed 13 (52%), where 8 (32%) and 5 (20%) were moderate and weak biofilm producers, respectively. Significant correlation was showed between A. hydrophila isolates and different cultivation temperatures. This clarified the potential virulence of A. hydrophila isolates from both fish and human sources and their public health hazard.