Here, 70 potential Vibrio pathogens belonging to nine species, dominated by Vibrio harveyi, were isolated and identified from diseased aquacultured marine fish in South China. Subsequently, the prevalence of 11 virulence genes and the resistance to 15 antibiotics in these strains were determined. Most strains possessed atypical virulence genes in addition to typical virulence genes. Notably, hflk and chiA originating from V. harveyi, and flaC associated with V. anguillarum were detected in more than 40% of atypical host strains. Multidrug resistance was widespread: 64.29% strains were resistant to more than three antibiotics, and the multi-antibiotic resistance index ranged from 0.00 to 0.60. The proportions of strains resistant to the antibiotics vancomycin, amoxicillin, midecamycin, and furazolidone all exceeded 50%; nevertheless, all strains were sensitive to florfenicol, norfloxacin, and ciprofloxacin. Furthermore, both virulence genes and antibiotic resistance were more prevalent in Hainan than in Guangdong, owing to the warmer climate and longer annual farming time in Hainan. These results therefore suggest that warming temperatures and overuse of antibiotics are probably enhancing antibiotic resistance and bacterial infection. This study reveals that pathogenic Vibrio spp. with multi-antibiotic resistance are highly prevalent among marine fish in South China and thus warrant further attention. The results will provide helpful guidance for ecological regulation and local antibiotic use in the control of marine fish farming' Vibrio diseases in South China, facilitating the implementation of national green and healthful aquaculture.