Owing to the richness of bioactive compounds, Olea europea leaf extracts exhibit a range of health effects. The present research evaluated the antibacterial and antiviral effect of leaf extracts obtained from Olea europea L. var. sativa (OESA) and Olea europea var. sylvestris (OESY) from Tunisia. LC-DAD-ESI-MS analysis allowed the identification of different compounds that contributed to the observed biological properties. Both OESA and OESY were active against Gram-positive bacteria (MIC values between 7.81 and 15.61 μg/mL and between 15.61 and 31.25 μg/mL against Staphylococcus aureus ATCC 6538 for OESY and OESA, respectively). The antiviral activity against the herpes simplex type 1 (HSV-1) was assessed on Vero cells. The results of cell viability indicated that Olea europea leaf extracts were not toxic to cultured Vero cells. The half maximal cytotoxic concentration (CC50) values for OESA and OESY were 0.2 mg/mL and 0.82 mg/mL, respectively. Furthermore, both a plaque reduction assay and viral entry assay were used to demonstrate the antiviral activity. In conclusion, Olea europea leaf extracts demonstrated a bacteriostatic effect, as well as remarkable antiviral activity, which could provide an alternative treatment against resistant strains.