Lepidium sativum is widely used as a culinary and medicinal herb and is claimed to cure many diseases. In this study, an attempt was made to investigate the biochemical composition and functional properties of L. sativum ethanolic extract. The extract contained a total phenolic content of 11.03 ± 0.75 (mg GAE/g dw plant material) and a flavonoid content of 4.79 ± 0.24 (mg QE/100 g dw plant material). Further, the extract was characterized by LC-ESI-Q-TOF-MS/MS profiling and the results showed that the ethanolic fraction contains many important phenolics such as Kaempferol, Coumaroylquinic acid, p-Coumaroyl glycolic acid, Caffeic acid. The identified compounds are known for their biological properties and therefore, the functional properties of the extract as a whole were also studied. The extract showed significant antioxidant activity (IC50 values) 162.4 ± 2.3, 35.29 ± 1.02, 187.12 ± 3.4 and 119.32 ± 1.5 μg/ml in terms of DPPH, ABTS, Superoxide scavenging activity and metal chelating property respectively. Further, the extract showed IC50 values, 73.72 ± 1.23 and 121.78 ± 1.03 μg/ml in HRBC membrane stabilization ability and protein denaturation inhibition capacity respectively, which in turn is a measure of its anti-inflammatory activity. The results of the study are promising and serve basis for further investigation into the plant and possible consideration for use in nutraceuticals and functional foods.