The essential oil of the leaves of Cupressus sempervirens L. was isolated by hydrodistillation and tested against gram positive and gram negative bacteria, showing remarkable antimicrobial activity against Bacillus subtilis with minimum inhibitory concentration (MIC) 75%. The antiviral activity of the essential oil was tested against Herpes simplex virus type 1 (HSV-1), showing antiviral activity with virucidal percentages of 68.0% and 53.2% at concentrations of 1:32 and 1:64, respectively. We firstly reported the isolation of two epi-betulin esters of fatty acids from the CHCl3 fraction of Cupressus sempervirens L. leaves, which were isolated and purified using HPLC, and identified using PMR and MS. The CHCl3 fraction showed significant cytotoxicity against HeLa cells.