Dandruff is a scalp disorder caused by a fungus (Malassezia sp.). Water spinach (Ipomoea aquatica) contains phenolic compounds which have antifungal activity. The purpose of this research is to know the active fraction of water spinach (Ipomoea aquatica) herbs that have an anti-dandruff activity against Malassezia sp. The study used a true experimental design for an anti-dandruff activity test. Fractionation of water spinach (Ipomoea aquatica) methanol extracts used column chromatography which stationary phase with silica gel 60 powder and mobile phase with chloroform: ethanol: acetyl acetate (8:2:0,1). In vitro anti-dandruff activity based on minimum inhibitory concentration (MIC) and minimum antifungal concentration (MFC) against Malassezia sp. of human dandruff isolate. The average of MIC and MFC among groups fraction of water spinach (Ipomoea aquatica) methanol extracts compared used Friedman test (p≤ 0,05). Fractionation produces 12 fractions, and fraction number 7 of water spinach (Ipomoea aquatica) herbs have the best anti-dandruff activity against Malassezia sp. with MIC 125 μg/mL and MFC 250 μg/mL identified as a phenolic compound. Based on the results, a fraction of phenolic compounds from water spinach (Ipomoea aquatica) herbs have anti-dandruff activity against Malassezia sp.