Protection of olive cultivars, Olea europaea L., from diseases and the development of more sophisticated control methods are indispensable for a renovated and competitive olive sector. In this context, the volatiles obtained by the main Tunisian oil cultivar Chemlali and both the introduced cultivars Arbequina and Koroneiki were tested for their antimicrobial activity against several dangerous pathogens by diffusion and dilution methods (in 2014). To evaluate the adaptation to biotic stress, the antioxidant potential was additionally evaluated. The volatiles extracted from leaves, stems and flowers of the tested cultivars exhibited interesting antimicrobial and antioxidant activities, reaching in many cases 100% of inhibition. To identify the bioactive compounds, GC-FID and GC-MS were performed, permitting to identify up to 97.8% of total compounds. Both non-terpene hydrocarbons and terpenes were present in important proportions among volatiles.