Endophytic actinomycetes isolated from Datura stramonium L. was evaluated for its effects against in vitro alpha-glucosidase inhibition, antioxidant, and free radical scavenging activities. Based on microbial cultural characteristic and 16S rRNA sequencing, it was identified as Streptomyces sp. loyola UGC. The methanolic extract of endophytic actinomycetes (MeEA) shows remarkable inhibition of alpha-glucosidase (IC50 730.21 +/- A 1.33 mu g/ml), scavenging activity on 2,2-diphenyl-picrylhydrazyl (DPPH) (IC50 435.31 +/- A 1.79 mu g/ml), hydroxyl radical (IC50 350.21 +/- A 1.02 mu g/ml), nitric oxide scavenging (IC50 800.12 +/- A 1.05 mu g/ml), superoxide anion radical (IC50 220.31 +/- A 1.47 mu g/ml), as well as a high and dose-dependent reducing power. The MeEA also showed a strong suppressive effect on rat liver lipid peroxidation. Antioxidants of beta-carotene linoleate model system revels significantly lower than BHA. The total phenolic content of the extract was 176 mg of catechol equivalents/gram extract. Perusal of this study indicates MeEA can be used as natural resource of alpha-glucosidase inhibitor and antioxidants.