Digera muricata Mart, a plant having therapeutic characteristics that has been utilised traditionally, belongs to the Amaranthaceae family, and a promising source of specific natural products utilized as antioxidant, prophylactic, antimicrobial, anthelmintic, anti-diabetic, and allelopathic agent. In the present study, a biologically active phytosterol was isolated from Digera muricata Mart. The isolated compound was characterized by 13C, 1H NMR, FTIR, and HRMS. Characterization of the isolate was done by antimicrobial assay, and molecular docking. The antimicrobial potential of the isolated phytosterol (50 µl) against Streptococcus pyogenes was found to be maximum (ZOI-20.0 ± 1.0), followed by Streptococcus agalactiae (ZOI-11.3±1.5), Candida albicans (ZOI- 09.0 ± 1.0), Klebsiella pneumonia (ZOI-8.6 ± 1.5) and Escherichia coli (ZOI-8.6 ± 1.5). The molecular docking results indicate that the phytosterol binds to the receptor 1AI9 at the 32^th and 58^th positions; 1KZN receptor at the 76^th position, the 5L3J receptor at the 46^th (ASN) and 136^th (ARG) position; 7WIJ receptor at the 419^th (ARG) and 582^th (ASP) and 585^th (ASN) positions.