Diphenyl diselenide, (PhSe)2 , is an organoselenium compound with pharmacological actions mostly related to antioxidant and anti-inflammatory properties. The study investigated its antiviral and virucidal actions against herpes simplex virus 2 (HSV-2) infection in vitro and in a vaginal infection model in mice. The plaque reduction assay indicated that (PhSe)2 showed virucidal and antiviral actions reducing infectivity in 70.8% and 47%, respectively. The antiviral action of (PhSe)2 against HSV-2 vaginal infection was performed by infecting mice (10(5) PFU/ml(-1) ) at day 6. The treatment with (PhSe)2 (5 mg/kg/day, intragastric [i.g.]) followed 5 days before and for more 5 days after infection. The extravaginal lesion score was evaluated from days 6 to 10. At day 11, animals were killed, and histological evaluation, determination of viral load, and TNF-α and IFN-γ levels were performed in supernatants of homogenized vaginal tissue. The levels of reactive species (RS), protein carbonyl, non-protein thiols (NPSH), nitrate/nitrite (NOx), and malondialdehyde (MDA), and the activities of myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were determined. (PhSe)2 reduced the histological damage, extravaginal lesion scores, the viral load of vaginal tissue, and the activity of MPO, but increased the levels of TNF-α, IFN-γ. (PhSe)2 attenuated the increase of RS, MDA, NOx levels and the activity of GR caused by infection. (PhSe)2 also attenuated the reduction of NPSH content and the inhibition of CAT, SOD, and GPx activities. The antiviral action of (PhSe)2 against HSV-2 infection was related to its immunomodulatory, antioxidant, and anti-inflammatory properties. J. Cell. Biochem. 117: 1638-1648, 2016. © 2015 Wiley Periodicals, Inc.