The antiviral activity was tested of different polarity extracts, with differing chemical composition, obtained from aerial parts of Heterophyllaea pustulata Hook f. (Rubiaceae) against Herpes Simplex Virus Type I (HSV-1) and Saint Louis Encephalitis Virus (SLEV). The Vero cell line was employed as a host cell for the antiviral assessment of benzene (Ben), ethyl acetate (EtOAc) and ethanol (EtOH) extracts by means of the Neutral Red uptake assay and plaque reduction test. None of the extracts showed antiviral activity against SLEV. Only the extracts (Ben and EtOAc) with a high content of anthraquinones (AQs) inhibited HSV-1 replication, exhibiting Selectivity Index (SI) values of 2.7 and 2.4, respectively. Therefore, these extracts could be good candidates as natural sources for antiviral drug development against HSV-1.