C. glabrata has become a prominent pathogen less susceptible to azole antifungal agents when compared with other Candida spp. Aims Our work aimed to study some virulence factors of C. glabrata isolates and to test the activity of Chemlali olive leaf extract (OLE) against virulence and the survival of clinical C. glabrata isolates. Methods The virulence factors of a collection culture of C. glabrata isolated from blood culture (n = 35), vulvovaginal (n = 21), and buccal site (n = 5) were analyzed in specific medium with and without OLE. Results All C. glabrata isolates tested were able to produce haemolysin and to form biofilm (n = 61), 98.3% of isolates were phospholipase producer and 96.7% were protease producer. No significant difference between Pz-values was obtained from oral cavity, vulvovaginal and blood culture isolates (P-value ≥ 0.05). C. glabrata growth was dramatically decreased after treatment with olive leaves extract and exhibited MIC90 value in the range of 2–8 mg/mL and MFC value in the range of 16–32 mg/mL. There was a significant (P Conclusion C. glabrata isolates from Sfax University Hospital presented a high virulent power and these activities do not depend on the sampling sites but on possible evolutionary changes that may occur between different regions of the world. Olive leaves extract evaluated here is a potential therapeutic drug for the treatment of C. glabrata infection not only in the growth but also in inhibition of virulence factors.