The cultivation of a <i>Streptomyces</i> sp. SD53 strain isolated from the gut of the silkworm <i>Bombyx mori</i> produced two macrolactam natural products, piceamycin (<b>1</b>) and bombyxamycin C (<b>2</b>). The planar structures of <b>1</b> and <b>2</b> were identified by a combination of NMR, MS, and UV spectroscopic analyses. The absolute configurations were assigned based on chemical and chromatographic methods as well as ECD calculations. A new chromatography-based experimental method for determining the configurations of stereogenic centers β to nitrogen atoms in macrolactams was established and successfully applied in this report. These compounds exhibited significant bioactivities against the silkworm entomopathogen <i>Bacillus thuringiensis</i> and various human pathogens as well as human cancer cell lines. In particular, piceamycin potently inhibited <i>Salmonella enterica</i> and <i>Proteus hauseri</i> with MIC values of 0.083 μg/mL and 0.025 μg/mL, respectively. The biosynthetic pathway involved in the formation of the cyclopentenone moiety in piceamycin is discussed.