Bacteria are the most prevalent cause of morbidity and mortality among wild fishes. Vibrio alginolyticus (V. alginolyticus) is one of the most serious fish pathogens. This study was performed to investigate prevalence of V. alginolyticus infection among six species of Red Sea fishes at Hurghada city, Egypt through clinical and bacteriological examinations and to determine pathogenicity of the isolates, their antimicrobial susceptibilities and to determine the minimum inhibitory concentration (MIC) of silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs) against V. alginolyticus. Therefore, a total number of 180 fish from these six fish species (30 fish of each species) were randomly collected from the Red Sea at Hurghada city during the period from October 2019 to March 2020 and subjected to clinical and bacteriological examinations. Based on the morphological and biochemical characteristics of the isolates, 64 (35.6%) homogenous V. alginolyticus isolates were isolated and phenotypically identified from the examined fishes with prevalence of (26.7%), (20%), (63.3%), (36.7%), (23.3%) and (43.3%) among Lutjanus ehrenbergii (L. ehrenbergii), Lethrinus borbonicus (L. borbonicus), Siganus rivulatus (S. rivulatus), Rhabdosargus haffara (R. haffara), Scarus ghobban (S. ghobban) and Cheilinus lunulatus (C. lunulatus) fishes respectively. Afterthat, V. alginolyticus was cofirmed as V. alginolyticus by 16S rRNA gene sequence and phylogenetic analysis and it was deposited in GenBank as V. alginolyticus MR-C17 (GenBank accession no. MW790239). The V. alginolyticus infected fishes exhibited skin dark coloration, scales loss, hemorrhages on several parts of the body surface, mouth, base of fins.