Frequency of active human herpesvirus-6, -7 (HHV-6, HHV-7) and parvovirus B19 (B19) infection/coinfection and its association with clinical course of ME/CFS was evaluated. 108 ME/CFS patients and 90 practically healthy persons were enrolled in the study. Viral genomic sequences were detected by PCR, virus-specific antibodies and cytokine levels—by ELISA, HHV-6 variants—by restriction analysis. Active viral infection including concurrent infection was found in 64.8% (70/108) of patients and in 13.3% (12/90) of practically healthy persons. Increase in peripheral blood leukocyte DNA HHV-6 load as well as in proinflammatory cytokines' levels was detected in patients during active viral infection. Definite relationship was observed between active betaherpesvirus infection and subfebrility, lymphadenopathy and malaise after exertion, and between active B19 infection and multijoint pain. Neuropsychological disturbances were detected in all patients. The manifestation of symptoms was of more frequent occurrence in patients with concurrent infection. The high rate of active HHV-6, HHV-7 and B19 infection/coinfection with the simultaneous increase in plasma proinflammatory cytokines' level as well as the association between active viral infection and distinctive types of clinical symptoms shows necessity of simultaneous study of these viral infections for identification of possible subsets of ME/CFS.