Pumpkin (Cucurbita moschata) contains bioactive compounds that are spread almost throughout the organs. One of the important contents of pumpkin is seed oil which has antioxidant activity. Antioxidants play a role in counteracting free radicals so that it is widely used to improve health quality. Fruit storage can affect seed quality and antioxidant activity in it. The purpose of this study was to evaluate and compare the chemical characteristics and antioxidant activity of pumpkin seed oil with different fruit storage times. The method for obtaining pumpkin seed oil from fruit storage for 10, 20, and 30 Days After Anthesis (DAH) was carried out using the soxhlet extraction method. The extracted oil was thickened using a rotary evaporator. Furthermore, its chemical characteristics were tested including yield, acid number, peroxide number, saponification number, water content. Pumpkin seed oil was measured for its antioxidant activity using the DPPH method. Absorbance was measured at a wavelength of 526 nm using a UV-Vis spectrophotometer. The data obtained were analyzed using ANOVA followed by the DMRT test with a 95% confidence interval. The results showed that there were significant differences in the variation of fruit storage time including oil yield, acid number, saponification number, and antioxidant activity, while there was no difference in water content. Fruit storage time of 30 DAH gave the most optimal results in all parameters tested. The conclusion that can be drawn from this study is the characteristics and antioxidant activity of pumpkin seed oil at a storage time of 30 DAH produced better oil quality compared to storage times of 10 and 20 DAH.