Bacterial immunity against antibiotics causes increased mortality. In dealing with this problem, studies have been carried out, one of which is by utilizing natural medicinal materials such as Dioscorea pyrifolia Kunth. tuber. Dioscorea pyrifolia is one of the medicinal plants that have not been widely studied so that in this study antibacterial activity testing will be carried out, the mechanism of action against leakage bacterial cells and component analysis of chemical extracts. Research carried out includes the collection and identification of plants, extraction, determination of antibacterial activity and analysis of chemical compounds using Thin Layer Chromatography; determination of the mechanism of action using an ultraviolet spectrophotometer, atomic absorption spectrometer and scanning electron microscopy. The results of research that have been carried out are known to extract ethyl acetate and methanol which have an inhibitory diameter as antibacterial activity against Escherichia coli and Staphylococcus aureus. The extraction method that produces the strongest antibacterial activity is the multilevel percolation method using ethylacetate as a solvent. The chemical compounds found in ethyl acetate extract are glycosides, alkaloids and polyphenol compounds, such as tannins and flavonoids. The mechanism of bacterial cell leakage caused by Dioscorea pyrifolia tuber extract can be proven by ultraviolet spectrophotometry, atomic absorption spectrophotometer and scanning Electron microscope. © RASĀYAN. All rights reserved.