Six undescribed sesquiterpenoids, including three acorane-type sesquiterpenoids (daphneaines A-C), three guaiane-type sesquiterpenoids (daphneaines E-G) and three known analogues were isolated from the roots of Daphne genkwa Siebold et Zucc. Their gross structures were elucidated by comprehensive spectroscopic analyses. The relative configurations of daphneaines A-C were determined by NOESY experiments. In addition, the relative configuration of daphneaine G was elucidated by performing a quantum chemical calculation of the NMR chemical shifts coupled with an advanced statistical procedure DP4+. The comparison of experimental and calculated electronic circular dichroism (ECD) data led to the establishment of the absolute configurations of daphneaines A-G. All isolates were tested for their inhibitory activity towards the LPS-induced NO production in RAW 264.7 macrophages, and daphneaine F showed inhibitory effect on NO production with an IC value of 35.68 ± 3.18 μM.