Aims: To determine the antiviral activities of Spirogyra spp. algal extracts against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). Methods and Results: Spirogyra spp. was extracted using water, ethanol and methanol. Aqueous extract of Spirogyra spp. had the lowest toxicity on Vero cells with the 50% cytotoxicity concentration (CC50) of 4363.30 mu g ml(-1). As for potent inhibitory effect, the ethanolic extract presented the highest inhibition of viral infection on HSV-1 in the treatment during viral attachment on Vero cells with 50% inhibitory concentration (IC50) and selective index (SI) values of 164.20 and 2.17 mu g ml(-1). However, the methanolic extract showed the highest inhibition of HSV-2 when treated during viral attachment with IC50 and SI values of 75.03 and 3.34 mu g ml(-1). The methanolic extract of Spirogyra spp. also demonstrated significant virucidal effects on viral particles. Therefore, anti-HSV activity at various stages of the viral multiplication cycle was shown. The main active compounds in the active fractions of Spirogyra spp. ethanolic extract against HSV were found to be alkaloids, essential oils and terpenoids. Conclusions: The highest anti-HSV activity was obtained from the ethanolic extract of Spirogyra spp. The extract inhibited the HSV viral particles and the inhibition was during the viral attachment and the viral multiplication. Significance and Impact of the Study: Anti-HSV activity of extract of freshwater green macroalga Spirogyra spp. in Thailand was demonstrated. Therefore, anti-HSV product containing the Spirogyra spp. extract should be developed for treatment of HSV infection.