Infectious diseases are a major problem in the health sector in Indonesia. One cause is the bacteria Escherichia coli. Uncontrolled use of antibiotics lead to increased bacterial resistance to antibiotics. Traditional crops needed to solve the problem proficiency level. Biduri leaves believed to be used as an antibacterial. The purpose of this study is to investigate the antibacterial activity of leaf extracts biduri (Calotropis gigantea L.) against Escherichia coli. Biduri leaves extracted using 70% methanol by maceration. This research used Completely Randomized Design (CRD) was divided into six treatment groups, consisting of the positive control (chloramphenicol 30 mcg), negative control Dimethyl sulfoxide (DMSO) 10% and biduri leaf methanol extract at a concentration of 25%, 50%, 75% and 100% repeated 4 times. Data were analyzed using Analysis of Variance (ANOVA) followed by Least Significant Difference test (LSD). The results showed that the methanol extract of the leaves biduri have antibacterial activity in concentration of 25%, 50%, 75%, and 100% with an average inhibition zone 6.12 mm, 6.75 mm, 7.62 mm and 8.75 mm. The results of ANOVA and LSD (p