Two new polycyclic tetramate macrolactams, lysobacteramides A (1) and B (2), together with HSAF (heat-stable antifungal factor, 3), 3-dehydroxy HSAF (4), and alteramide A (5) were isolated from a culture of Lysobacter enzymogenes C3 in nutrient yeast glycerol medium. Their structures were determined by MS and extensive NMR analysis. The absolute configurations of 1-5 were assigned by theoretical calculations of their ECD spectra. Although HSAF and analogues were reported from several microorganisms, their absolute configurations had not been established. The isolation and the absolute configurations of these compounds revealed new insights into the biosynthetic mechanism for formation of the polycycles. Compounds 1-4 exhibited cytotoxic activity against human carcinoma A549, HepG2, and MCF-7 cells with IC50 values ranging from 0.26 to 10.3 μM. Compounds 2 and 3 showed antifungal activity against Fusarium verticillioides with IC50 value of 47.9 and 6.90 μg/mL, respectively.