Human herpesviruses (HHVs) are involved in the pathogenesis of different types of uveitis. Cytokine response plays an important role in virus-induced immunopathology. This study aimed to investigate the incidence of HHVs in aqueous humor samples of immunocompetent patients with suspected viral uveitis and cytokine (IL-10, IL-6, and IFN-γ) expression profiling. Forty-seven aqueous humor samples were collected from immunocompetent patients with viral uveitis. Samples were assayed for HHV-1 to HHV-8 by in-house real-time polymerase chain reactions. IL-6, IL-10, and IFN-γ were quantified with a cytometric bead array. Relations between viral detection, cytokine profiles, and clinical data were studied. At least one viral genome was detected in 21 aqueous humor samples analyzed. Varicella-zoster virus (VZV) was detected in 14 of the positive samples, cytomegalovirus (CMV) in 8, HSV-1 in 1, Epstein-Barr virus (EBV) in 4, and HHV-6 in 2. More than one viral genome was detected in seven aqueous humor samples. Aqueous humor samples positive for HHV-DNA contained significant levels of IL-6, IL-10, and IFN-γ, compared to HHV-DNA negative samples. High levels of IL-6 were detected in patients with CMV-DNA in their aqueous humor samples. Significantly higher levels of IL-10 and IFN-γ were found in positive samples for VZV, EBV, and HHV-6 DNA than in negative aqueous humor ones. VZV was the principal etiologic agent of uveitis in this Tunisian series, with CMV the second most common agent. Knowledge of immunoregulatory interactions and dynamic changes in viral uveitis may be a key to understand the pathogenesis leading to more-effective treatments.