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A fungus, Aureobasidium pullulans, was isolated from marine biofilm and identified. A bioassay-guided fractionation procedure was developed to isolate and purify antifouling compounds from A. pullulans HN. The procedure was: fermentation broth—aeration and addition of sodium thiosulfate–graduated pH and liquid–liquid extraction—SPE purification—GC–MS analysis. Firstly, the fermentation broth was tested for its toxicity. Then it was treated with aeration and addition of sodium thiosulfate, and its toxicity was almost not changed. Lastly, antifouling compounds were extracted at different pH, the extract had high toxicity at pH 2 but almost no toxicity at pH 10, which suggested the toxicants should be fatty acids. The EC50 of the extract against Skeletonema costatum was 90.9μgml−1, and its LC50 against Balanus amphitrete larvae was 22.2μgml−1. After purified by HLB SPE column, the EC50 of the extract against S. costatum was 49.4μgml−1. The myristic and palmitic acids were found as the main toxicants by GC–MS.

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