Safe and effective nonsteroidal anti-inflammatory drugs are needed. Meanwhile, addition of amino acids to cultures of microorganisms is likely to increase the possibility of novel secondary metabolite isolation. In the course of screening for anti-inflammatory agents using cellular lipopolysaccharide (LPS)-induced nitric oxide (NO) production, two new related compounds with the myceliothermophin structure from a methionine-enriched culture of Myceliophthora thermophila ATCC 42464 were isolated. The new compounds have an additional methylthio group on the myceliothermophin structure and were named myceliostatins A and B. Both compounds inhibited LPS-induced NO production at nontoxic concentrations in macrophage-like mouse monocytic leukemia RAW264.7 cells. Myceliostatin B inhibited the expression of LPS-induced iNOS, IL-6, and IL-1β and the upstream NF-κB activity in situ and in vitro. Finally, it was found to inhibit NF-κB binding to DNA in the reconstruction system with purified p65. Myceliostatin B also inhibited LPS-induced reactive oxygen species (ROS) production. Thus, myceliostatin B, a novel compound derived from M. thermophila, was found to be a new anti-inflammatory and antioxidant compound directly inhibiting NF-κB.