Berberis aristata is used as a traditional medicine in India to accelerate the process of wound healing. Comparative antimicrobial activity of different samples obtained from the stem and root of Berberis aristata was evaluated against two Gram-negative bacteria (Salmonella enterica and Shigella flexneri) and a lipolytic yeast (Candida aaseri). The extraction was done using hot continuous extraction and maceration methods to compare the concentration of berberine and to evaluate the antimicrobial activity by the well diffusion method. The results showed that the hydroalcoholic root extract obtained by the hot continuous extraction process possessed a higher concentration of berberine than other extracts. Moreover, the hydro-alcoholic root extract was also found most effective against microbial strains. The study suggested that berberine might be responsible for the antimicrobial activity of the extract of Berberis aristata.