The inhibitory activity of extract and compounds isolated from Boesenbergia longiflora against nitric oxide (NO) was evaluated using RAW264.7 cells. Isolation of the chloroform extract of B. longiflora rhizomes afforded four known flavonoids, which were identified as kaempferol-3,7,4'-trimethyl ether (1), kaempferol-7,4'-dimethyl ether (2), rhamnazin (3), pinostrobin (4), together with four known diarylheptanoids, dihydrobisdemethoxycurcumin (5), curcumin (6), demethoxycurcumin (7) and bisdemethoxycurcumin (8), as well as one sterol, -sitosterol-D-glucoside (9). Compound 6 exhibited the highest inhibitory activity against NO release with an IC50 value of 4.5 μM, followed by 7 (IC50 = 11.7 μM), 8 (IC50 = 15.7 μM), 5 (IC50 = 23.0 μM) and 1 (IC50 = 23.5 μM), respectively. This study demonstrated that diarylheptanoids and some methoxyflavonoidsfound in B. longiflora are responsible for anti-inflammatory activity and this is the first report the safety, chemicalconstituents and biological activity of this plant.