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BACKGROUND Quassinoids and canthin-6-one alkaloids are bioactive markers of Eurycoma longifolia (EL) and E. harmandiana (EH) and have been commercially utilized to treat inflammation and male infertility. OBJECTIVES This study aims to reveal the contents of bioactive compounds and to compare anti-inflammatory activities of these two species. METHODS High-performance liquid chromatography (HPLC) methods coupled with UV-Vis detection were developed and validated for the simultaneous analysis of the chemical profiles and their contents in EL and EH. The anti-inflammatory activities of both species were investigated using RAW 264.7 cell line. RESULTS The HPLC methods provided a sensitivity (LOD) of 0.02-0.05 µg/mL for the eight bioactive compounds (canthin-6-one alkaloids, quassinoids, and scopoletin) with high precision (% relative standard deviation (RSD) ≤6.48) and recoveries between 80.0-120%. The chaparrinone: eurycomanone ratio was high in EH, whereas EL had a higher ratio of eurycomanone: chaparrinone than EH. The contents of total canthin-6-one alkaloids, quassinoids, and scopoletin were 0.01-0.75, 0.19-1.54, and 0.01-0.28 mg/g, respectively, in EL roots and 0.12-1.80, 7.05-9.26, and 0.02 mg/g, respectively, in EH roots. The anti-inflammatory effects of EL and EH extracts varied among the samples due to the variation in their chemical constituents. CONCLUSIONS In summary, our study indicated that chaparrinone was the major compound in EH. EH exhibited anti-inflammatory activity the same extent as EL. HIGHLIGHTS EH and EL extracts were analyzed using developed HPLC-UV methods, revealing a high concentration of chaparrinone in EH, and an anti-inflammatory assay indicated that EH had a potency comparable to that of EL.