Martynia annua L. is a local herb often used by the people of Timor Island as a traditional medicine. This study aims to determine the types of secondary metabolite compounds and antibacterial activity of M. annua flower extract against the growth of Escherichia coli Mig. Extraction was carried out by the maceration method using 96% ethanol. Antibacterial tests were carried out by the disc diffusion method, with treatment concentrations of 200 ppm, 400 ppm, 600 ppm, 800 ppm, 1000 ppm, tetracycline positive control, and negative control using distilled water. Data were analyzed using analysis of variance. The results showed that M. annua flower extract contains alkaloids, flavonoids, steroids, and saponins. Antibacterial activity tests showed that M. annua flower extract inhibited the growth of E. coli, with an effective concentration of 400 ppm, an average inhibition zone of 10.36 mm, and was classified as strong. Thus, this plant has potential for future use, particularly in relation to the development of medicinal plants. However, further research is needed to utilize and commercialize this plant product.