Nitric oxide (NO) plays an important role in the physiology and pathophysiology of the central nervous, cardiovascular, and immune systems. Inducible nitric oxide synthase (iNOS) produces higher levels of NO which is implicated in the pathogenesis of various inflammatory diseases such as septic shock, rheumatoid arthritis, inflammatory bowel disease, and neurodegenerative diseases. Activation of microglia is a histopathological hallmark of neurodegenerative diseases and is believed to contribute to neurodegenerative processes through the release of various pro-inflammatory cytokines and the overproduction of NO. An inhibitor of NO production in microglia cells may be a potential therapeutic agent for intervention of various inflammatory and neurodegenerative diseases. In the course of screening for inhibitors of NO production in BV-2 microglia cells, a new potent compound named trienomycin G (1) and the related known compound trienomycin A (2) were isolated from a fermentation broth of Streptomyces sp. 91614. We report here the fermentation, isolation, physico-chemical properties, structure determination, and biological activities of 1. The producing strain was isolated from a soil sample in Korea and assigned to Streptomyces sp. Fermentation, extraction, and purification steps yielded 1 as a white powder. The structure of 1, determined via spectral data including 1H/13C NMR, HMQC, HMBC, and NOESY, is a structural isomer of 2 differing in the linkage position of the N-hexahydrobenzoylalanine moiety (linked to C-13 in 1 instead of C-11 in 2). Both 1 and 2 exhibited potent inhibitory effect on NO production in LPS-stimulated BV-2 microglia cells with EC50 values of 292.3 and 25.4 nM, respectively. They showed cytotoxic activity with IC50 values of 6274 and 1244 nM, respectively, which were much higher than their inhibitory activity, suggesting the inhibitory effect on NO production is not due to cell viability loss.