Objective: To ascertain the antimicrobial activity of methanolic leaf extracts of Justicia adhatoda and vasicine against Staphylococcus aureus, Streptococcus pyogenes, Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Cryptococcus neoformans and Aspergillus flavus. Methods: The antimicrobial activity of the concentrated leaf extracts of J. adhatoda was evaluated by determination of the diameter of zone of inhibition against bacteria and fungi. 25μg ml-1 concentration was used to check the antimicrobial activity of plant extracts and vasicine. Minimum inhibitory concentrations and minimum microbicidal concentrations were determined against all the pathogens. Sensitivity of the pathogens was also checked with four standard antibiotics, ciprofloxacin and ofloxacin for bacteria and nystatin and amphotericin B for fungi. Results: The phytochemical studies revealed the presence of alkaloids in the extracts were active against both bacteria and fungi. Studies on the minimum inhibitory concentration of the extracts on the test organisms showed that the lowest minimum inhibitory concentration and minimum microbicidal concentrations were demonstrated against Serratia marcescens, Escherichia coli and Pseudomonas aeruginosa and the highest minimum inhibitory concentration was exhibited against Staphylococcus aureus, Streptococcuspyogenes, Klebsiella pnuemoniae. Among fungi Aspergillus flavus showed lowest minimum inhibitory concentration whereas Candida albicans and Cryptococcus neoformans showed highest minimum inhibitory concentration. Conclusion: The present study revealed that J. adhatoda has broad spectrum of antimicrobial activity and a potential source of antimicrobial agents that could be useful for chemotherapy and control of infectious diseases.