Although Garden cress (Lepidium sativum) seeds are commonly used in Amhara region, studies on the chemical constituents of these seed varieties are lacking in the literature. In this study, the composition of total Phenolic content (TPC) and flavonoids content (TFC); and their free radical scavenging activity of Garden cress seed varieties collected from Fogera woreda (South Gondar zone), Yilmana Densa woreda (west Gojjam zone) and Lay Armachiho woreda (North Gondar zone) were studied. The two varieties (red and black colored) garden cress seeds collected from each sampling sites were extracted using methanol (100%) and aqueous methanol (80%) with ultrasound assisted extractor. Qualitative phytochemical screening test for the extracts were carried out for the presence of phytochemicals: flavonoid, Triterpene, steroids, glycosides, tannins, alkaloids, and saponins. Each extracts were investigated colorimetrically for TPC, TFC and antioxidative activities using UV-VIS Spectrophotometer and results were expressed as gallic acid equivalent (GAE), Catechin equivalent (CE) and Ascorbic acid equivalent (AAE) per gram of sample, respectively for TPC, TFC and anti-oxidant activity. The maximum antioxidant activity, total Flavonoid and total phenolic content were obtained from 80% aqueous methanol extract as compared to 100% methanol. These differences were significant at (P<0.05).It was found that the antioxidant activity, total Flavonoid and total phenolic content of the aqueous methanol extract of the seed varieties ranged from 35.03±3.84 to 46.66±1.43 mg AAE/g, 5.93±0.39 to 8.47±1.29 mg CE/g and 10.55±0.80 to12.66±0.70 mg GAE/g of sample, respectively. In this study, black colored seed sample from Yilmana Densa had the highest content of total phenolic and antioxidant activity than other seed samples and also black seed sample from Fogera had the maximum total Flavonoid content as compared to other varieties. Sample from Lay Armachiho (red seed type) had the lowest antioxidant, total Flavonoid and total phenolic content than other varieties and districts. Nutshell black seeds have the highest phytochemicals and antoxidant activity than the red variety.