Berkeleylactone A is a potent 16-membered macrolactone antibiotic, recently isolated from a coculture of Berkeley Pit Lake fungi. Although its antimicrobial activity has already been investigated, little is known about the structure-activity relationship. Based on our previous synthetic studies, a series of berkeleylactone A derivatives were synthesized and evaluated for their in vitro antimicrobial activities against methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA) strains. Our data confirmed the essential role of the embedded conjugated system and suggest a reversible sulfa-protection of the Michael acceptor as a viable option. Structurally simplified achiral macrolactam 8 showed the best inhibitory activity against S. aureus L12 (MRSA) with MIC50 values of 0.39 μg mL-1, 8-fold lower than those of berkeleylactone A. These studies may be of value in the development of more advanced candidates for antibiotic applications.