In this paper, the disinfection of water is investigated using plasma spark treatment and the results are compared with conventional techniques. Inactivation of the Enterococcus faecalis and Escherichia coli bacteria is considered in the treatment process of water by the plasma spark. For this purpose, many physical and chemical parameters of water are measured and the obtained results demonstrate a reduction of 8-log in colony forming units of E. coli and E. faecalis at 15 minutes and 12 minutes, respectively. The results of this research show that no ozone is produced during the plasma spark treatment. Moreover, inactivation of a large number of bacteria without any change of pH shows that pH is not the cause of the bacterial inactivation. It is concluded that the main causes of the inactivation of bacteria in the treated water are H2O2 molecules and the electrical fields generated by plasma.