The development of strains of herpes simplex virus (HSV) resistant to drugs has been reported among the immunocompromised patients. Thus, there is a need to develop new therapeutic agents for HSV infections. We evaluated the anti-HSV activity of Bidens pilosa (B. pilosa), a tropical weed, in tissue culture cells and a mouse model. B. pilosa extract showed potent virucidal activity. It inhibited plaque formation and suppressed virus yield in Vero and RAW 264.7 cells infected with HSV-1 and HSV-2. Both the binding of virus to host cells and penetration of virus into cells were also blocked by B. pilosa. Furthermore, B. pilosa was effective against thymidine kinase-deficient and phosphonoacetate-resistant HSV-1 strains. B. pilosa treatment increased the survival rate of HSV-infected mice and limited the development of skin lesions. Our results indicate that B. pilosa has anti-HSV activity and is thus a potentially useful medical plant for treatment of HSV infection.