Edwardsiella tarda is a common fish pathogen, causing septicemic diseases and formidable economic losses in freshwater fish farming. This study aimed to investigate the prevalence, molecular typing, antibiogram pattern, pathogenicity, distribution of virulence determinant genes (edw1, cds1, qseC, and pvsA) and antibiotic resistance genes (blaTEM, blaCTX, sul1, tetA, qnrA, qnrB, qnrS, and aadA1) associated with natural E. tarda infection among Nile tilapia (Oreochromis niloticus) and African catfish (Clarias gariepinus). Thus, 200 fish (100 Nile tilapia and 100 catfish) were collected randomly from different localities in Dakahlia Governorate, Egypt and subsequently processed for clinical and bacteriological examinations. E. tarda was recovered from 24 fish, with the highest prevalence being reported in C. gariepinus (14%) followed by O. niloticus (10%). The liver and kidney were affected mostly with a prevalence of 54.5% and 36.4%, respectively. PCR results revealed that all the tested isolates harbored gyrB conserved gene, edw1 and qseC virulence genes, while 77.2% were positive for cds1gene. Paradoxically, none of the tested strains were positive for pvsA gene. Based on antibiogram and molecular analysis of resistance genes, most of the tested isolates were multi-drug resistance to six antimicrobial classes (penicillins, aminoglycosides, fluoroquinolones, tetracyclines, sulfonamides, and lincosamides) and harbored blaTEM, sul1, tetA, blaCTX-M, aadA1, qnrS, and qnrA resistance genes as well as gryB, edwI, qseC, and cds1 virulence genes. The challenged fish exhibited similar clinical signs and postmortem lesions found in the naturally infected fish. Moreover, the mortality rate was 65% and 40% in C. gariepinus and O. niloticus, respectively. Therefore, the emergence of multi-drug resistant isolates of E. tarda represents a threat to fish farming sector in Egypt. Molecular-based detection is the fundamental tool for precise and early detection of E. tarda and for controlling septicemic diseases in aquaculture.