Five new phenolic glycosides, tenuisides A-E (1-5), and a new megastigmane glycoside, tenuiside F (6), along with seventeen known compounds (7-23) were isolated from the aerial parts of Polygala tenuifolia Willd. Their structures were established by detailed analysis of NMR and HRESIMS spectroscopic data, and the absolute configurations of compounds 5 and 6 were determined by CD spectra and in-NMR-tube Mosher's method. The inhibitory effects of these compounds were evaluated on NO production in LPS-activated BV-2 microglia cells. Compound 17 showed the strongest activity, with an IC50 value of 7.4μM, while compounds 1, 8, 14, and 18 showed the moderate activities, with IC50 values of 16.2-38.5μM. And their primary structure-activity relationships (SARs) of NO inhibitory effects were also briefly discussed.