Enteroviruses, especially Coxsackie B viruses (CBVs), are responsible for approximately 50% of cases of viral myocarditis. In the present study, serum samples (160) were collected from acute myocarditis patients at different age groups and 104 samples of the same age groups as a control. Cholesterol, LDH, CPK, and GOT were measured for all serum samples (264). Also, to study the source of virus transmission, 72 water and 72 wastewater samples were collected from water and wastewater treatment plants at intakes and outlets. Water and wastewater samples were concentrated by filtration through Zeta-plus filter cartridges and reconcentrated by the PEG-6000 precipitation method. Serum, water, and wastewater samples were inoculated in BGM cells for three successive passages. RT-PCR with enterovirus primers was carried out directly for serum samples and for 1st and 3rd cell culture passages. The positive samples were used for neutralization assay using anti-CBV sera pool to determine the CBV followed by neutralization with separate antisera. The results showed that 50 (31.25%) serum samples from acute myocarditis patients and two (1.4%) samples from the controls were positive for enterovirus RT-PCR. For water and wastewater samples enteroviruses were present in 63.8% and 8.3% for intake and outlet of water treatment plants and, 66.6% and 47.2% for intake and outlet of wastewater treatment plants, respectively. The level of CBV serotypes was varied where CBV3 was dominant for all age groups of myocarditis patients and CBV2 and CBV5 were also detected while CBV2 was the main CBV in water samples and CBV2, 3 and 5 were detected in wastewater samples. The integration of cell culture-PCR reduces the time required for virus detection and enhances the sensitivity of the test.