The Avocado leaves are one part of the avocado plant that has an antibacterial activity such as, inhibition of Escherichia coli bacteria. Normally, Escherichia coli live in the lower gastrointestinal tract and to become pathogenic when the amount and growth of germs in the body exceeds the normal limits. The aims of the study to determine the antibacterial activity of n-hexane fraction of avocado ethanolic leaf extracts against to the growth of Escherichia coli bacteria trough in vitro test. The extraction process used was maceration for 3 days and re-maseration for 2 days followed by fractionation process using n-hexane as the solvent. Qualitative analysis of the alkaloid compounds in the extract was done by using dragendroff test and yielded orange precipitate. The TLC test was performed to identify the compounds contained in the n-hexane non-polar fraction. The n-hexane fraction was then made into four concentrations (12.5%, 25%, 50% and 100%) as the samples of antibacterial activity test by using the Kirby-Bauer or Disk Diffusion method. The TLC tests showed the presence of alkaloid compounds in the n-hexane fraction by Rf values of 0.225 and 0.725 with orange blotches. The results of this study show that the nonpolar n-hexane fraction of avocado ethanolic leaf extracts (Persea americana Mill.) is able to inhibit the growth of Escherichia coli bacteria at the lowest level of 12.5% to the highest level of 100% with the lowest average DZI value of 7.67 mm and highest is 11,67 mm.