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Roots of <i>Rondeletia odorata</i> are a rich source of phytochemicals with high antioxidant potential and thus may possess health benefits. This study used the LC-MS technique to identify phytoconstituents in <i>R. odorata</i> roots extract/fractions. Results revealed that <i>n</i>-butanol fraction and ethanolic extract contained total phenolic and flavonoid contents with values of 155.64 ± 0.66 mgGAE/g DE and 194.94 ± 0.98 mgQE/g DE, respectively. Significant potential of antioxidants was observed by DPPH, CUPRAC and FRAP methods while the ABTS method showed moderate antioxidant potential. Maximum % inhibition for urease, tyrosinase and carbonic anhydrase was shown by ethanolic extract (73.39 ± 1.11%), <i>n</i>-butanol soluble fraction (80.26 ± 1.59%) and ethyl acetate soluble fraction (76.50 ± 0.67%) which were comparable with thiourea (standard) (98.07 ± 0.74%), kojic acid (standard) (98.59 ± 0.92%) and acetazolamide (standard) (95.51 ± 1.29%), respectively, while all other extract/fractions showed moderate inhibition activity against these three enzymes. Hemolytic activity was also observed to range from 18.80 ± 0.42 to 3.48 ± 0.69% using the standard (triton X-100) method. In total, 28 and 20 compounds were identified tentatively by LC-MS analysis of ethanolic extract and <i>n</i>-butanol soluble fraction, respectively. Furthermore, molecular docking was undertaken for major compounds identified by LC-MS for determining binding affinity between enzymes (urease, tyrosinase and carbonic anhydrase) and ligands. It was concluded that active phytochemicals were present in roots of <i>R. odorata</i> with potential for multiple pharmacological applications and as a latent source of pharmaceutically important compounds. This should be further explored to isolate important constituents that could be used in treating different diseases.