Olive tree is one of the most important fruit trees because of its content of many potentially bioactive compounds and represent waste products from olive harvest and pruning of olive trees. However, the content of these compounds can differ between cultivars. The aim of the current study was to evaluate the antioxidant, antimicrobial and anti-inflammatory properties of three olive leaves cultivars (Picual, Shemlali and Tofahy) commonly cultivated in Egypt. Furthermore, determine their cytotoxicity for safety use in food applications. Three olive leaves cultivars were extracted with water and yield of extract was determined in each cultivar, the antioxidant activities, anti-inflammatory, cytotoxic and antimicrobial activity of 7 pathogenic bacteria, 1 yeast and 2 fungi species were determined. Of all the extracts tested, Picual had the highest content of each of phenolic (TPC) and flavonoid (114.79±4.19 µg GAE/mg,118.69±2.07 µg CE/g extract, respectively) and exhibited strong antioxidant potential in scavenging DPPH. Antibacterial potential method was against Escherichia coli ATCC25922, Escherichia coli BA12296,Klebsiella pneumonia ATCC12296, Salmonella spp., Staphylococcusaureus EMCC1351, Streptococcus mutans EMCC1815, Bacilluscereus EMCC1006, ClostridiumperfringensEMCC1574. Antifungal activities of: Aspergillusniger EMCC72, AspergillusparasiticusEMCC886T and Candidaalbicans EMCC15, were evaluated and the minimum inhibitory concentration (MIC) of all cultivars values for bacteria and yeast ranged from 12.5 to 100 mg/ml. The extracts from Picual, Tofahy and Shemlali of 5µg/ml had the highest anti-inflammatory effect. Cytotoxicity results indicated high safety use of the three olive leaves cultivars. Theresults confirmed convenient and safe use of extracts of three olive leaves cultivars as natural antimicrobial and antioxidant.