The aim of current investigation was to study the lipids composition and biological activities of fixed oil extracted from D. lotus root. The lipids composition of the recovered oil from D. lotus roots including fatty acid methyl esters (FAME), along with squalene were determined by gas chromatographic/mass spectrometery (GC/MS). The main constituents of oil were linoleic acid (44.9%), linoleic acid (17.8%), palmitic acid (10.8%), squalane (3%), oleic acid (2.97%), stearic acid (2.38%), and palmitic acid (2.20%). The oil was screened for antimicrobial, antioxidant, and cytotoxicity activities. D. lotus oil was subjected to various biological activities like antimicrobial, antioxidant, cytotoxicity, and insecticidal assays. D. lotus oil exhibited antimicrobial traits against Straph epidermis, Klebsiello pneumonia and Bacillus subtilis with zone of inhibition ranging from 12-15 mm. D. lotus crude oil did not show insecticidal activity against Tribolium castaneum, Rhyzopertha dominica and Callosbruchuanalis. D. lotus oil showed brine shrimp cytotoxic effect at woo mu g/ml dose with LD50 2997.99 as compared to etoposide LD50 7.4625. The degree of lethality was directly proportional to the concentration of D. lotus oil. The reults showed that D. lotus oil revealed considerable antimicrobial and antioxidant activities. The oil could be applied in different pharmaceutical and nutraceutical applications.