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The antimicrobial activity of endophytic fungi isolated from Euphorbia milii was evaluated against Gram-positive, Gram-negative bacteria, unicellular yeast, and filamentous fungi. Chaetomium ovatoascomatis NRC was identified morphologically and genetically as the most active strain. The total ethyl acetate extract of C. ovatoascomatis NRC demonstrated significant antimicrobial activity against Gram-negative; Escherichia coli, Salmonella enteric, and fungi; Aspergillus niger with MIC of 62.5 ug/ml. Whereas n-hexane fraction demonstrated broader activity against Gram-positive; Bacillus subtilis, Lactobacillus cereus, Gram-negative; Escherichia coli and Salmonella enteric, fungi; Candida albicans and F. solani. LC–MS/MS analysis of ethyl acetate strain extract and GC–MS analysis of the n-hexane fraction were used to identify the metabolites of the strain extract. LC–MS/MS determined three major metabolites with potential antimicrobial activities including grevilline B, aflatoxin G2 and apigenin. GC–MS analysis of n-hexane fraction tentatively identified 30 compounds, where 9,12-octadecadienoic acid methyl ester was the major compound.

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