Five novel naturally occurring diacetylenic spiroacetal enol ethers, named chracetylespienoles A-E (1-5), along with five known analogues (6-10), were isolated from the aerial part of Chrysanthemum indicum. A combination of extensive spectroscopy, chemical derivatization, DP4+ NMR analysis, and ECD calculations was employed to determine the new compounds' structures and assign their absolute configurations. The anti-inflammatory activity of 1-10 was determined by measuring their suppression on NO production in lipopolysaccharide (LPS)-mediated RAW 264.7 cells, and 8 showed the best NO production inhibitory activity with an IC₅₀ value of 7.42 ± 0.87 μM. Furthermore, compound 8 downregulated the expression of iNOS and COX-2 proteins by inhibiting the MAPK and activating the Nrf2 signaling pathways, thereby suppressing inflammatory cascades.