Abstract This study aimed to investigate the effect of Ultrasound-Assisted Extraction (USAE) and Microwave-Assisted Extraction (MWAE) methods on the quality parameters and yield of silkworm pupae oil. The selected key independent variables that impact the extraction process, such as power levels, liquid-solid (L/S) ratio, and time for the extraction of SPO (silkworm pupae oil), were optimised. The extracted silkworm pupa oil was further characterised for oil quality, thermal behaviour using DSC (Differential Scanning Calorimeter), colour values and fatty acid profile for its suitability for various food applications. USAE resulted in a maximum oil yield of 36.60 ± 0.20% at 400 W with an L/S ratio of 6 ml/g at 20 min pulsation time. The fatty acid profile of SPO through GC-MS revealed significant amounts of linoleic acid and linolenic acid (6.50% and 67.18%) in USAE, followed by MWAE (34.40 ± 0.20%). Further, the FTIR spectra revealed high-intensity bands ranging between 2300-3000 cm−1, associated with functional groups from long-chain alkyl groups and triglycerides. It was observed that SPO had Omega-3 and Omega-6 long chain fatty acids and triglycerides which are essential for human health. Antioxidant activity exhibited high free radical scavenging in USAE (27.46 ± 0.15%) followed by MWAE (26.63 ± 0.15%). The morphology of SEM images of USAE exhibits microstructural damage to cells resulting in the release of more oil. DSC thermograph displayed that crystallisation and melting curve temperatures for USAE and MWAE were lower than SXLE (soxhlet extraction) alone due to higher amounts of unsaturated fatty acids. SPO extracted using USAE exhibited higher oil yield with significant antioxidant capacity and good thermal properties for its application in food products.