A novel C17 homo-guaiane sesquiterpene (1) and twenty-nine new guaiane-type sesquiterpenes (5–18, 23–25, 27–30, and 33–40), along with ten known analogues were isolated from Daphne penicillata. Compound 1 may be derived from an intermolecular aldol reaction and represent the first natural product containing an additional methyl acetate (C3). Their structures were determined by the analysis of spectroscopic data, quantum-chemical calculations, and single crystal X-ray crystallographic analysis. Interestingly, a detailed spectroscopic analysis of 2–26 indicated that characteristic Cotton effects (CEs) and the chemical shift of H-7 were associated with C-1 configuration, leading to a simple and convenient method for the rapid assignment of C-1 configuration in this family of guaiane-type sesquiterpenes. The underlying mechanism was further discussed by conformational and molecular orbital analyses. Moreover, all isolates were evaluated for their inhibitory activity against lipopolysaccharide-induced NO production in murine microglia BV2 cells.