The consequence of water stress on the polyphenolic content, anticholinesterasic and antifungal activities of methanolic leaf extracts of three Tunisian (Olea europea L.) varieties: Meski, Ouslati, and Jarbouii was investigated. Our results showed that total polyphenolic and flavonoids compounds amplified in all tested varieties under water stress. In addition, water stress increased the anticholinesterasic activity in all all olive cultivars. But Oueslati variety had the best one with the lowest IC50. Antifungal activity of all extracts of olive leaf generally increased under water deficit. We can conclude that water stress exposed positive effect on secondary metabolites; hence the biological effect of olive leaf of different varieties. We can conclude that water stress induced an increase in biological activities (antifungal and anticholinesterasic effects). In addition, Ouslati cultivar may be considered as the most tolerant one, followed by Jarboui and Meski was the most sensitive. Ouslati cultivar may be considered as the most tolerant one, followed by Jarboui and Meski was the most sensitive.