The aim of this study was to carry out HPTLC (high-performance thin-layer chromatography) fingerprinting analysis and to identify by LC/MS (liquid chromatography– mass spectrometry) the main components of a hydroethanolic extract of cypress (Cupressus sempervirensL.) fresh cones; the study also aimed at evaluating the extract’s virucidal activity against four human viruses and three bovine viruses. The phytochemical analysis showed the presence of tannins, flavonoids, amino acids, and carbohydrates. At 40% (v/v) concentration, in contact with viruses for 60 minutes at 37 °C, the extract had virucidal activity against coronavirus, parainfluenza type 3 virus, bovine respiratory syncytial virus, and bovine rotavirus. At 80% (v/v), the extract had virucidal activity against influenza A-H1N1 virus, rhinovirus, and bovine herpes type 1 virus. These results show that it would be worthwhile to pursue research on this cypress extract and to explore its antiviral effects in vivo.