The quantity and quality of olive oil have been affected by various factors such as climatic and regional conditions, cultivar, and harvest time. This study was conducted to evaluate the effect of different harvesting times on the oil quantity, quality, and profile of fatty acids in four cultivars of olive, including Roghani, Zard, Amphissis, and Beladi in Isfahan region. For this purpose, the olive fruits were harvested at four stages (two weeks intervals) starting in early October. At each stage of harvesting, the quantitative and qualitative characteristics of the oil were evaluated and the profile of fatty acids was examined. The results showed that in all cultivars, with the progress of fruit ripening and increasing the maturity index, the percentage of fruit oil and the free fatty acid increased and chlorophyll content and phenolic compounds in the oil decreased. In this study, the lowest amount of saturated fatty acids (palmitic acid and stearic acid), the highest ratio of monounsaturated fatty acids (oleic acid) to polyunsaturated fatty acids (linoleic acid), and the lowest rate of cox index (oxidation index) that indicate higher quality of olive oil were obtained in the second stage of harvest time in Amphissis and Roghani and in the fourth stage of harvest time in Zard and Beladi cultivars. According to the results of this study, in all cultivars the highest oil content were obtained when fruits harvested at the latest time (November 14).