Background: Due to frequent exposure to surface water and contact with animals, children represent a group susceptible to zoonotic diseases. Objectives: The present study aims to determine the presence and prevalence of the main zoonotic agents in R. norvegicus populations in Tehran, Iran. Methods: In the present study, 100 R. norvegicus were captured within a time span of one year from five districts of Tehran, Iran. Fecal and blood samples were collected from rodents and serum was recovered after centrifugation. The presence of specific IgG antibodies against Leptospira spp. and Rabies virus was detected using a commercial qualitative rat ELISA kit. A conventional PCR assay was employed to detect the presence of Vibrio vulnificus in the commensal R. norvegicus population. Results: In general, 80% (n = 80/100) and 20% (n = 20/100) of rats were males and females, respectively. The results of the ELSA assay showed that of the 100 R. norvegicus captured in Tehran, 7% (n = 7/100) and 1% (n = 1/100) were positive for Leptospira spp. and Rabies virus, respectively. Leptospira spp. revealed the highest frequency (20%; 4/20) among R. norvegicus collected from the eastern part of Tehran. Rabies virus was detected only from the southern (5%; 1/20) part of Tehran. Results of the PCR method showed that the percentage of the rats tested positive for V. vulnificus was 5%. Overall, the surveyed zoonotic microorganisms had the highest (n = 5/20; 25%) and lowest (n = 1/20; 5%) frequency rates in the eastern and northern parts of Tehran, respectively. Conclusions: The results accentuate the necessity of implementing rodent control programs and regular disinfection as well as avoiding contact with rodent populations in urban environments.