<p>Pumpkin seed oil (<i>Curcubita mochata</i>) consists mainly of unsaturated fatty acids such as oleic acid and linoleic acid. Extraction of pumpkin seed oil while retaining its high nutritional quality is a challenge, as conventional solvents are not suitable for the extraction. In this study, the parameters of ultrasound-assisted extraction (UAE) of pumpkin seed oil were optimised using ethanol and hexane as solvents and compared to two conventionally used extraction techniques (Soxhlet and Bligh and Dyer). An experimental design with a response surface and a two-level 2² factorial approach with 5 replicates at the central point for two solvents (ethanol and hexane) was performed. 2 variables were investigated: (%) amplitude and temperature (°C). The optimal conditions for extraction with hexane and ethanol were as follows: 44.48% and 69.25% amplitude, 69.19 °C and 50 °C, with a yield of 25.054% and 25.01%, respectively. A coefficient of variation of less than 5% was obtained, indicating that the predicted performance is quite homogeneous compared to the validated performance. The analysis of the fatty acid profile of the pumpkin seed oil revealed a significant content of unsaturated fatty acids (80% consisting of oleic acid and linoleic acid), with significant differences between conditions and extraction methods. The comparison among the methods showed that UAE was a more efficient method with a significant concentration of oleic acid and linoleic acid for a possible further eco-friendly technology.</p>