Introduction: The phytochemical analysis, antimicrobial activity, and in vitro antidiabetic assay of extracts obtained from the seeds of mature Indian Dudhi plant also known as Milk hedge (Euphorbia nivulia) were examined in this study. Materials and Methods: Using aqueous methanol extracts of the seeds of E. nivulia, phytochemical components were investigated. Further using chromatography, different components were separated and its antimicrobial activity was investigated. Results and Discussion: Two components from the seed extract were isolated using column chromatography. Four different types of bacterial cultures were utilized for evaluating the antimicrobial activity. The results indicated that seed extracts are more sensitive toward Gram-negative organisms. Conclusion: Thus, the presence of vial phytochemicals in the seed extract could render it therapeutic potential which can be utilized for the treatment of different diseases including microbial infections.