As a part of our bioassay-guided isolation from Boesenbergia longiflora rhizomes, it is indicated that the chloroform (CHCl3) fraction possessed potent anti-inflammatory activity. Therefore, the CHCl3 fraction was then separated by chromatographic method which finally obtained three new sesquiterpenes (1–3) together with eleven known compounds (4–14). Their structures were elucidated by spectroscopic and X-ray crystallographic methods. The present study is aimed to investigate the isolated compounds from Boesenbergia longiflora rhizomes as well as the anti-inflammatory activity using RAW264.7 macrophage cells and experimental animals.