Medicinal plants are usually screened for phytochemicals that may lead to further isolation,purification, and characterization of their active principles. The roots of Rumex abyssinicus are usedtraditionally in Ethiopia for treating different diseases such as hepatitis, hemorrhoids, gonorrhea,typhus, and wounds. Thus, this study was conducted to investigate the phytochemicals and in vitroantimicrobial activity of the roots of R. abyssinicus extracts. The roots were collected, chopped, and airdried under shade in the laboratory. Then, the dried plant material was subjected to sequentialextraction using the maceration technique by soaking 1 kg of the sample in petroleum ether,chloroform, acetone, and methanol. Meanwhile, the filtrates were concentrated under reduced pressureusing a rotary evaporator. Afterward, the crude extracts were dried and screened for in vitroantimicrobial activity tests using four bacterial and two fungal strains. Based on its activity, the acetoneextract was used for column chromatographic isolation. Herein, two known anthraquinones wereisolated and characterized: chrysophanol and emodin. The microbial activity of both extracts andisolated compounds were evaluated against medically important strains by the agar disc diffusionmethod. The concentration of the extracts and isolated compounds was 100 mg/mL tested against S.aureus ATCC25903, K. pneumoniae NCTC13368, E. coli ATCC 25722, P. aeruginosa DSMZ 1117 and C.albicans and S. cerevisiae. The zone of growth inhibition of extracts and isolated compounds werecompared with standard drugs like Gentamycin and Mancozeb. The results showed that the isolatedcompounds (1 and 2) have higher bacterial growth inhibition than the extracts. However, compound 2was not active against S. cerevisiae strain.