Oil extracted from various species of silkworm pupae emerged as an alternative source of edible oil with great therapeutic value. However, the Tasar silkworm, is mainly known for producing more textured silk than the applications of its pupal oil. The current study aimed to extract characterize and investigate edible and therapeutic properties of Tasar Pupal Oil (TPO).TPO was extracted by Soxhlet extraction method. Characterization of TPO was conducted by GC/MS and FTIR. The Total phenolic content of TPO was evaluated by Folin-Ciocalteu assay. The safety evaluation of TPO was checked on healthy HEK293 (human embryonic kidney cells) using MTT assay. Whereas, therapeutic properties of TPO were analyzed through DPPH, FRAP, SRB assay and agar-well diffusion method. GC/MS study revealed that α-linolenic acid (ALA) (38%) was the major component of TPO followed by n-hexadecanoic acid (25.64%), and other minor compounds were detected in small amounts. The functional groups of TPO shown by FTIR analysis correspond with similar peaks of edible oils. The TPO was reported to be low in phenolic content. The DPPH and FRAP radical scavenging assays demonstrate that TPO exhibits significant antioxidant capabilities. Further, TPO revealed a substantial dose-dependent anticancer activity against colon cancer cells, with a GI50 value of ≤20 μg/ml. Interestingly, TPO was safe and non-toxic on HEK293 cells. Further, TPO exhibited antibacterial activity against S. typhi and S. mutans at various dilutions. Our findings clearly reveal the potentiality of TPO by displaying the characteristics of edible oil and therapeutic properties including antioxidant, anticancer, and antibacterial.