Objective: The present study was undertaken to evaluate the antimicrobial activity of an ointment prepared from alcohol, ethyl acetate, and chloroform extracts of Eupatorium glandulosum Hort. ex Kunth. Methods: The dried plant materials of E. glandulosum were individually extracted using alcohol, ethyl acetate, and chloroform by Soxhlet method. These three different types of extracts were used for the preparation of ointment and evaluated for the phytochemicals, pH, homogeneity, spreadability, tube extrudability, stability, and antimicrobial activity. The activity of prepared ointments was evaluated against Escherichia coli and Bacillus subtilis and the in vitro antimicrobial activity of ointments of plant extracts was performed by cup plate methods. The most effective antimicrobial ointment was determined by comparing the results of the zone of inhibition of all the ointments of each solvent extract. Results: The antimicrobial activity of optimized formulation was showed significant activity against the tested bacterial pathogens. Conclusion: The present study confirmed the antimicrobial activity of the formulations. Furthermore, it can use as a potential dosage form for clinical utility. The results are the justification for the use of the plant in folk medicine.