Medicinal plants remain an important source of antimicrobial agents, particularly in regions where traditional remedies are widely practiced. Croton macrostachyus Hochst ex. Delile (Euphorbiaceae) is commonly used in Ethiopian traditional medicine to manage infectious conditions; however, its bioactive constituents and antibacterial mechanisms are not fully understood.This study aimed to identify major phytochemical constituents from the roots of C. macrostachyus and to evaluate their antibacterial efficacy using experimental and computational approaches. Root extracts were subjected to silica gel column chromatography, resulting in the isolation of four previously reported compounds. The antibacterial activity of both the crude extracts and purified compounds was tested in vitro against four reference bacterial strains of human pathogens. In addition, molecular docking simulations were carried out to predict the binding interactions of the isolated compounds with Escherichia coli DNA gyrase B (PDB ID: 6F86) and Staphylococcus aureus Sortase A (PDB ID: 1T2P). Chemical investigation of the root material yielded lupeol, β-sitosterol, stigmasterol, and linoleic acid. Antibacterial assays showed that the extracts and isolated compounds inhibited the growth of the tested pathogens to varying extents. Docking analysis demonstrated stable binding of the compounds within the active sites of both target proteins, with calculated binding energies between −7.38 and −5.57 kcal/mol for DNA gyrase B and −7.40 and −5.54 kcal/mol for Sortase A. The combined experimental and in silico findings suggest that C. macrostachyus roots contain bioactive metabolites with antibacterial potential. These results provide mechanistic support for the traditional application of this plant in Ethiopia for the management of infectious diseases, including skin and respiratory ailments.
