Endogenous olive enzymes represent a crucial link between fruit composition and the nutritional and sensory characteristics of virgin olive oil. Enzymes such as β-glucosidase, polyphenol oxidase and peroxidase influence the content and composition of polyphenols, the main antioxidants in virgin olive oils. Lipoxygenase and hydroperoxide lyase convert fatty acids into volatile components responsible for the specific sensory properties of the oil. The aim of the present study was to determine the influence of fruit ripeness on the enzymatic activity of β-glucosidase, polyphenol oxidase, peroxidase and lipoxygenase in four Croatian autochthonous olive cultivars. For this experiment, olive fruits from two Dalmatian (Oblica and Levantinka) and two Istrian cultivars (Rosulja and Istarska bjelica) grown in their area of origin were harvested on three different harvest dates, from October 28 to November 9, 2021. The β-glucosidase, polyphenol oxidase and peroxidase were isolated from the acetone powder of the paste. The activity of these enzymes was determined spectrophotometrically, and the formation of p-nitrophenol from p-nitrophenyl glucopyranoside was measured for β-glucosidase, the formation of o-quinone from catechol for polyphenol oxidase, and the formation of tetraguaiacol from guaiacol for peroxidase. Lipoxygenase was isolated directly from olive paste, and its activity was determined by measuring the concentration of formed hydroperoxides by HPLC. The activity of β-glucosidase was detected only in a few samples, which was not sufficient to determine the influence of the factors studied on its activity. Polyphenol oxidase activity increased between the first and second harvest dates, and this increase was significantly higher in the Istrian cultivars. At later harvest dates, the activity decreased, but to a greater extent in the Dalmatian varieties. Peroxidase activity was significantly affected by both ripening and cultivar. Istrian cultivars had significantly higher peroxidase activity than Dalmatian cultivars. The activity was lower in riper fruits, with the exception of Levantinka, which had the highest activity in ripe fruits. Lipoxygenase activity was influenced primarily by harvest date: on the first date, activity was highest in Rosulja and Levantinka, on the second date in Istarska bjelica, and on the third date in Oblica. Throughout the harvest period, lipoxygenase activity was higher in Rosulja and Levantinka than in the other two cultivars.