Plesiomonas shigelloides (P. shigelloides), a dominant pathogenic strain identified as YY001, was successfully isolated and characterized from the hepatopancreas of diseased Mastacembelus armatus (M. armatus) for the first time. The strain was identified through a comprehensive approach that included morphological, physiological, and biochemical characterization, as well as 16S rRNA, 23S rRNA, and gyrB gene sequence analysis. Strain YY001 is classified as a Gram-negative bacterium, with optimal growth conditions observed at 28°C and pH 7.0. Results from the bacterium hemolysis test indicated the absence of hemolytic activity in YY001. However, the strain exhibited amplification of nine virulence genes: nanE, waaA, clpP, ssb, actP, aphA, astA, astD, and astB. Drug-resistance testing showed that YY001 displayed resistance only to penicillin G, oxacillin, norfloxacin, and tetracycline, while remaining sensitive to the majority of other antibiotics. Furthermore, nine drug-resistance genes, including emrD, catB, macB, ksgA, tolC, bacA, MdfA, sul1, and DegP, were detected through amplification. Experimental infection demonstrated that YY001 induced symptoms such as abdominal swelling, visceral edema, and hemorrhage, with hepatopancreatic manifestations being particularly severe. The median lethal dosage of strain YY001 in M. armatus was 9.7×108 colony-forming units (CFU)/mL within 48 h, and 4.9×106 CFU/mL within 192 h under the conditions of intraperitoneal injection. The hepatopancreas, gill, and intestine of infected fish showed obvious pathological symptoms, including tissue loosening, hemorrhage, and cell necrosis. The results of this study provided evidence for a more comprehensive understanding of P. shigelloides strain YY001 infection in M. armatus.