Global silk production generates about 1 million tons of spent pupae annually that are generally discarded as waste but could be used to generate about 240 million liters of biodiesel every year. Spent pupae contain about 30–40% oil, 30–40% proteins and about 20–30% carbohydrates which comprise a low‐cost, renewable and sustainable source for production of biofuels, particularly biodiesel. Some studies indicate the potential of converting the pupa oil into biodiesel but there are no reports on the detailed characteristics and the properties and performance of the pupa biodiesel. In this study, oil was obtained from the pupae using mechanical and Soxhlet extraction with yield of up to 95%. The oil was transesterified using acid and alkaline catalysts under different processing conditions. A conversion ratio of more than 90% from oil to biodiesel was possible. Pupae diesel had characteristics similar to regular synthetic‐based diesel with calorific value of between 44 41 MJ kg−1. Pupae diesel was blended up to 50% with regular diesel and performance of the pure and blended fuel was tested on an engine test rig. Engine performance tests showed that the pupa diesel did not compromise the efficiency and produced emissions within the stipulated standards. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd.