The bioactive phytochemical constituents present in the plant play a significant role in the development of medicines and drug discovery. Rumex abyssinicus is one of the plants that grow in Ethiopia and different parts of this plant are used as a traditional medicine. The aim of this research was isolation and characterization of bioactive compounds from Rumex abyssinicus rootbark. The plant material was collected from Oromia region around Ilu Ababor Zone, Metu College of teachers’ education campus during February 2016. The dried and powdered plant material was subjected to sequential solvent extraction using maceration technique to prepare the crude extract which was directly used for antibacterial studies. The lists of solvents taken for the extraction were n-hexane, chloroform, acetone and methanol. Antibacterial evaluation of crude extracts of the plant rootbark were screened using in vitro method against four pathogenic bacteria species namely Gram-positive Staphylococcus aureus, Gram negative Escherichia coli, Pseudomonas aeruginosa and Salmonella thyphimurium. These sequentially obtained solvent extracts were filtered and concentrated under reduced pressure. The results showed the acetone extract followed by methanol crude extracts showed inhibitory effects against all of the tested bacterial strains, but the isolated compounds showed inhibitory effects on S. aureus and P. aeruginosa bacterial strains. The acetone extract was subjected to column chromatographic separation that led to isolation of two compounds. The structures of these compounds were characterized with the help of spectroscopic methods (IR and NMR). The isolated compounds were characterized as diisobutyl phthalate (RA-2) (isolated for the first time from the plant) and Emodin (RA-3) based on spectroscopic data and in comparison, with literature reports.