Background: Today, people use plants to treat various types of diseases and improve human health. One of the medicinal plants is the Betadine plant (Jatropha multifida L.). Betadine plants have many functions, especially the sap, leaves, fruit and seeds. The compound contents in Betadine stem sap, which is efficacious as an antimicrobial, are saponins, tannins, flavonoids and labaditin. One of the bacteria that cause infection is Pseudomonas aeruginosa. These bacteria can cause opportunistic and nosocomial infections. Methods: This study was a true experimental laboratory with a post-test only control group design. This study used Betadine stem sap extract with concentrations of 25%, 50%, 75%, 100%, gentamicin cream 10% as positive control, and dimethyl sulfoxide (DMSO) solution as negative control. This study used the Kirby-Bauer diffusion method and the bacterium Pseudomonas aeruginosa was grown on nutrient agar media, then incubated for 24 hours and calculated using calipers. Research data were analyzed using one-way ANOVA test. Results: The highest inhibition zone was group 50% (12.725 ± 0.2500 mm) while the lowest inhibition zone was group 100% (8.675 ± 0.5620 mm). Conclusions: Betadine stem extract had antibacterial activity in inhibiting the growth of Pseudomonas aeruginosa bacteria, with the 50% concentration being the most effective in inhibiting the growth of Pseudomonas aeruginosa bacteria.