In the course of a searching environmental friendly antifungal compounds, we found that mixture of methyl esters of fatty acids obtained from soybean oil had potent control efficacy against barley powdery mildew (Blumeria graminis f. sp. hordei). In this study, ten alkyl fatty acid esters (AFAEs) were tested for in vivo antifungal activity against five plant diseases such as rice blast, rice sheath blight, tomato gray mold, tomato late blight and barley powdery mildew. Some AFAEs showed the most control efficacy against barley powdery mildew among the tested plant diseases. By 5-hr protective and 1-day curative applications, six AFAEs (3,000 μg/ml), including methyl and ethyl palmitates, methyl and ethyl oleates, methyl linoleate, and methyl linolenate demonstrated both curative and protective activities against barley powdery mildew. In contrary, methyl laurate strongly controlled the development of powdery mildew on barley plants by curative treatment at a concentration of 333 μg/ml, but did not show protective activity even at 3,000 μg/ml. Under greenhouse conditions, the seven AFAEs (1,000 μg/ml) except for methyl and ethyl stearates, and methyl caprylate also effectively controlled cucumber powdery mildew caused by Podosphaera xanthii. Among them, methyl and ethyl palmitates (333 μg/ml) represented the most control activity of more than 68% against the disease. The results are the first report on the antifungal activity of methyl and ethyl esters of fatty acids against plant pathogenic fungi.