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Cyclopia species are increasingly investigated as sources of phenolic compounds with potential as therapeutic agents. Recently, we demonstrated that a crude polyphenol-enriched organic fraction (CPEF) of Cyclopia intermedia, currently forming the bulk of commercial production, decreased lipid content in 3T3-L1 adipocytes and inhibited body weight gain in obese db/db mice. The aim of the present study was to determine whether a more effective product and/or one with higher specificity could be obtained by fractionation of the CPEF by purposely increasing xanthone and benzophenone levels. Fractionation of the CPEF using high performance counter-current chromatography (HPCCC) resulted in four fractions (F1–F4), predominantly containing iriflophenone-3-C-β-D-glucoside-4-O-β-D-glucoside (benzophenone: F1), hesperidin (flavanone: F2), mangiferin (xanthone: F3), and neoponcirin (flavone: F4), as quantified by high-performance liquid chromatography with diode array detection (HPLC-DAD), and confirmed by LC-DAD with mass spectrometric (MS) and tandem MS (MSE) detection. All fractions inhibited lipid accumulation in 3T3-L1 pre-adipocytes and decreased lipid content in mature 3T3-L1 adipocytes, although their effects were concentration-dependent. F1–F3 stimulated lipolysis in mature adipocytes. Treatment of mature adipocytes with F1 and F2 increased the messenger RNA expression of hormone sensitive lipase, while treatment with F1 and F4 increased uncoupling protein 3 expression. In conclusion, HPCCC resulted in fractions with different phenolic compounds and varying anti-obesity effects. The activities of fractions were lower than the CPEF; thus, fractionation did not enhance activity within a single fraction worthwhile for exploitation as a nutraceutical product, which illustrates the importance of considering synergistic effects in plant extracts.