With the anti-microbial and anti-tumor composite screening model, bioassay-guided fractionation led to the isolation of two structurally related bioactive compounds, curvularin and alpha beta-dehydrocurvularin, from ethyl acetate extract of Eupenicillium sp. associated with marine sponge Axinella sp. Further study on the structure-activity relationship demonstrated that both compounds exhibited differences in bioactive profiles which are highly associated with their minor structural differences. Both curvularin and alpha beta-dehydrocurvularin have similar level of anti-fungal and anti-tumorous activity, while alpha beta-dehydrocurvularin is active against Staphylococcus aureus with a minimal inhibitory concentration of 375 mu g/ml but curvularin does not. No detectable activity against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa exists for both compounds. It is suggested that the partial planar backbone structure, due to the conjugation of pi electrons in the presence of a 3,4-double bond and the carbonyl group at position C-2 in alpha beta-dehydrocurvularin, acts as a key factor for the inhibition of S. aureus, a Gram-positive low G + C bacteria that are often the hospital-acquired and/or community-acquired pathogen.