Vibrio cholerae is a gram-negative bacteria known for causing epidemics and pandemics conditions, comprised of both non-pathogenic and pathogenic strains. The present study identified a non O1, non-O139 V. cholerae (EMM1) serotype isolated from diseased Labeo rohita cultured in a wetland system at Purba Medinipur district, West Bengal, India. Identification of the isolate was done by biochemical test, 16S rRNA gene sequencing and phylogenetic analysis. The in vivo challenge experiment revealed that intraperitoneal injection of EMM1 serotype at a dose of 1.04× 107 CFU/ml resulted in 100% mortality of L. rohita. Furthermore, the presence of hlyA and rtxA virulent genes, confirmed by sequencing, in V. cholerae (EMM1) serotype might be, atleast partially involved in the mortality of L. rohita. In histological analysis, we found that EMM1 serotype challenge had a significant effect in L. rohita like degenerated hepatocytes in the liver, shrunken glomeruli in the kidney, necrosis of the intestinal villi epithelium and degeneration of muscle bundle were observed. Overall, the study demonstrated that, V. cholerae (EMM1) serotype is a potent aquatic microbial pathogen and draws the attention for proper management to prevent the possible occurrence of disease in aquaculture.