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Given the growing interest of consumers worldwide in safe, healthy and good quality olive oil, the present study aimed to investigate a number of Tunisian virgin olive oil varieties in an attempt to procure physico-chemical characteristics of interest to the local and global olive oil industry, particularly in terms of variety and area of cultivation. Eight samples collected from Chetoui, Chemlali, Zarrazi, and Zalmati Tunisian cultivars located at Sfax, Thibar, Zarzis, and Jerba were employed. Various physic-chemical parameters were investigated, including free acidity, peroxide value, UV absorption, pigment concentrations, oxidative stability, fatty acid composition, phenolic compounds and sterols. The findings revealed high heterogeneity within and between the varieties. The Chemlali Jerba" and Zarrazi oil samples showed high oleic acid and low linoleic acid concentrations, rich phenolic compounds, and high stability. The sterols fractions in Chemlali Sfax, Chemlali Jerba, and Zalmati olive oils displayed high levels of β-sitosterol, campesterol, and ∆-5-avenasterol. Overall, the olive oil from Chemlali Jerba presented higher levels of natural antioxidants and oxidative stability. Its chemical composition was also noted to be particularly interesting in terms of fatty acid contents, minor components, and antioxidants, namely sterols, tocopherols, carotenoids, and phenolic acids.