Listeria monocytogenes is a zoonotic food-borne disease with significant public health and economic consequences. In animals, L. monocytogenes can cause clinical listeriosis, which is characterized by symptoms such as abortion, encephalitis, and sepsis. In the current study, 100 random samples of fresh fish (Labeo rohita) were purchased from various fish forms and markets in Pakistan's Rawalpindi and Islamabad regions. L. monocytogenes was detected bacteriologically in the collected samples. Then virulence tests were applied to positive isolated L. monocytogenes samples, all of which showed positive Christie–Atkins–Munch-Peterson results, a narrow zone of hemolysis on sheep blood agar, and positive beta hemolytic activity. Five out of seven samples were detected positive for the presence of the virulence gene (hlyA) using the specific primers via conventional PCR technique. Labeo rohita had the highest prevalence of virulence genes. The results showed that 7.0% prevalence (7/100) of L. monocytogenes was obtained from fresh Labeo rohita samples identified by a biochemical test. All L. monocytogenes-positive isolates tested for antimicrobial susceptibility Some isolates showed resistance to ampicillin, tetracycline, ceftazidime, and azithromycin. They were, however, sensitive to chloramphenicol, gentamicin, levofloxacin, amikacin, and trimethoprim. Listeriosis zoonotic potential has been demonstrated in raw fish and open-air fish market samples, necessitating further monitoring and awareness of antibiotic resistance to identify contaminated foods and ensure effective treatment. Furthermore, the health authorities must establish more active food control and surveillance. Population groups at higher risk of listeriosis require preventive education programs.