From a further purification study, four new stemphones D to G were isolated along with previously reported stemphones B and C from the culture broth of Aspergillus sp. FKI-2136. Twenty-one derivatives were semisynthetically prepared from stemphones C, E and G. Potentiation of imipenem activity against methicillin-resistant Staphylococcus aureus (MRSA) by all the stemphones including natural and semisynthetic ones was compared to study the structure-activity relationships. Derivatives with a free hydroxy or an O-acyl residue having a C2 to C5 carbon length at C-4 held the potentiating activity, but those with a longer acyl residue lost the activity. The presence of an oxo or a free hydroxy residue at C-10 is important for the potentiating activity because introduction of an alkyl or acyl residue at this position resulted in a loss of activity. Among them, stemphone E exhibited the most potent potentiation of imipenem activity against MRSA and the lowest cytotoxic activity against Jurkat cells.