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Plants are good sources of various bioactive substances and have significant importance for the discovery of new drugs. In this study, Croton macrostachyus plant roots from six different sites in the Central Gondar Zone, Ethiopia, were collected; and their phenolic and flavonoid contents, as well as antioxidant and antibacterial activities were investigated. Total phenolics and flavonoid contents of the root extracts were determined by the Folin–Ciocalteu and aluminum chloride methods, respectively. The antioxidant activity was determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging method. Moreover, the antibacterial activities were evaluated by disk diffusion method. Results revealed that total polyphenols and flavonoid contents were in the range of 802 ± 53–1557 ± 75 mg GAE/100 g and 342 ± 26–745 ± 32 mg CE/100 g, respectively. Root extracts of C. macrostachyus were found to have higher antioxidant activities ranging from 3.53 ± 0.38 to 6.38 ± 0.62 mg AAE/g sample. They also showed inhibition zones of 5.8, 6.2, 5.9 and 6.0 mm for Staphylococcus aurous, Staphylococcus pneumonia, Escherichia coli and Klebsiella pneumonia, and had equivalent potency with the reference Gentamicin antibiotic. Pearson correlation result indicated a strong relationship between total polyphenol contents and their respective antioxidant activities. This study articulates that the root extracts accumulated a substantial quantity of polyphenols and bears a considerable antioxidant activity.