In our search for fungal metabolites with antibiotic activity against methicillin-resistant Staphylococcus aureus (MRSA), we have isolated three different setin antibiotics from several different fungi-the previously reported equisetin (2) and trichosetin (3), and a novel setin-like compound (1) [cissetin] from OSI 50185. Like the known setin antibiotics, cissetin (1) is active against several Gram-positive organisms, but it is most notable for the atypical cis ring fusion in the octalin portion of the molecule. Cissetin (1) was isolated as a white foamy powder; positive ion detection HR-FAB-MS suggested a molecular formula of C23H33NO4. In contrast to other setin class compounds, 1 gave crisp, clean 1H and 13C NMR spectra in CDCl3 at ambient temperature, allowing complete assignment of all resonances via COSY, HMQC, and HMBC. Deshielded octalin ring junction signals (H-6 and H-11) and their small coupling indicated cis stereochemistry at the octalin ring junction-previously unobserved in setin antibiotics-supported by comparison with muurola-4,11-diene (cis) and cadina-4,11-diene (trans). The saturated cyclohexane ring adopts a chair conformation, the cyclohexene ring a slightly twisted boat conformation, with stereochemical assignments confirmed by ROESY correlations. Biological activity assessment showed cissetin (1) had similar activity to equisetin (2) and trichosetin (3) against MRSA, MSSA, vancomycin-resistant Enterococcus faecium, E. faecalis, and was inactive against Escherichia coli. Cissetin (1) was 4-8 times more active than 2 and 3 against penicillin-resistant Streptococcus pneumoniae but completely inactive against Candida albicans (while 2 and 3 were marginally active). The similarity of antibiotic activities suggests the tetramic acid moiety confers the antibiotic qualities of the setins.