Despite considerable recent work to reveal different features of mushrooms species, the few studies of antiviral activities are inadequate and therefore further studies are required. Morchella conica, M. esculenta, Terfezia boudieri, Pleurotus ostreatus, Tricholoma anatolicum, Fomes fomentarius, Laetiporus sulphureus, Phellinus igniarius, Porodaedalea pini, and Pyrofomes demidoffii from Turkey were investigated to reveal their in vitro cytotoxic and anti-herpes simplex virus 1 (HSV-1) activities. The crude methanol extracts (MEs) and aqueous extracts (AEs) of fungal species and acyclovir (ACV) were used. Various dilutions were used to assess the cytotoxic effects of fungal species (50-0.10 mg/mL) and ACV (500-0.98 μg/mL) on uninfected Vero cells. Maximum nontoxic concentrations were determined for all extracts and ACV by comparing the optical densities of their cell controls. The concentration providing 50% protection against the cytopathic effect caused by the virus, extracts, and ACV (EC50) was calculated, and the half-maximal cytotoxic concentration (CC50) and the selectivity index (SI) were determined, the latter as the ratio of CC50 to EC50. While the AEs of F. fomentarius (EC50, 11.22 mg/mL; SI > 4.46), Ph. igniarius (EC50, 9.71 mg/mL; SI > 5.15), and P. pini (EC50, 7.16 mg/mL; SI > 6.98) showed considerable antiherpetic activity, MEs and AEs of the other fungal species did not showed any effects. The EC50 and SI of ACV were determined as 0.20 μg/mL and 3085, respectively. The results demonstrate that F. fomentarius, Ph. igniarius, and P. pini have important anti-HSV-1 activity.