Objective: The objective of this study was to discover of the ethanolic extract of Rhinacanthus nasutus (L) Kurz in inhibiting Escherichia coli bacteria using an in vitro method. Methods: This is an experimental study using a laboratory test with Kirby-Bauer or paper disc method by observing and measuring the inhibition zone of the ethanolic extract of R. nasutus against E. coli bacteria with extract concentrations of 15%, 30%, and 60% consisting of control groups and treatment group. The positive control group used chloramphenicol antibiotics and negative control groups used Aquadest. E. coli was incubated at 37°C for 24 h. Then, the plates were incubated for 24 h at 37°C and the diameter of the inhibition zone was observed until the 3rd day with three repetitions. Results: The results of the study showed that the mean inhibition zone of E. coli bacteria was 10.93 mm, 12.09 mm, and 18.90 mm. The results of the Shapiro–Wilk test were p=0.199. The results of the one-way analysis of variance test were p<0.05 and that of the post hoc test indicated a significant value of p<0.05. Based on the results of the research, there were significant differences in the inhibition zone between the control group and the treatment group at a concentration of 15%, 30%, and 60%. Conclusion: R. nasutus extract was effective to inhibit the growth of E. coli bacteria at concentrations of 15%, 30%, and 60%, so R. nasutus is effective as an antimicrobial.