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Aquaculture is one of the major economic activities in India, providing livelihoods and nutritional security to millions of people. In recent times, fish diseases have come to the limelight resulting in significant economic losses. We aimed to identify pathogenicity and virulence profiling of thirty-six pathogenic bacterial strains isolated from diseased Labeo rohita in the district of Hooghly, West Bengal, India. The bacterial strains were characterized through a comprehensive approach involving the examination of morphological features, biochemical properties, amplification, and sequencing of the 16S rRNA, species-specific genes, and virulence genes. Considering the prevalence frequency, virulence potential, and statistical significance Aeromonas hydrophila and Pseudomonas aeruginosa were selected for a survival assay followed by the examination of histopathological features to elucidate their effects. The identified bacterial isolates were arranged based on their predominance frequency, i.e., Aeromonas hydrophila (25%), Aeromonas veronii (22%), Pseudomonas aeruginosa (22%), Enterococcus faecalis (14%), Klebsiella pneumoniae (6%), Staphylococcus aureus (6%) and Escherichia coli (5%). Sixteen virulence-associated genes related to pathogenicity were amplified across the thirty-six isolates; aer, alt, lip and hlyA for A. hydrophila; exoS, lasB, toxA, oprL and phzM for P. aeruginosa; entB, fimH and uge in K. pneumoniae; aer in A. veronii; hlyA in E. coli; hlb in S. aureus and gelE for E. faecalis. The log-probit analysis revealed that A. hydrophila was notably more pathogenic than P. aeruginosa, as indicated by its lower lethal dose of 1.5×104 CFU/mL. Additionally, histological examination revealed notable pathological changes, including tissue degeneration, inflammatory cell infiltration and vacuolation observed in the liver, kidney, gill and intestine of the challenged fish. We highlighted several potent aquatic microbial pathogens in order to manage and prevent such aquacultural maladies.