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<i>Antrodia camphorata</i> is an endemic mushroom in Taiwan. This study was designed to screen anti-inflammatory compounds from the methanolic extract of the mycelium of <i>A. camphorata</i> on nitric oxide (NO) production in RAW 264.7 cells induced by polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA (dsRNA) known to be present in viral infection. A combination of bioactivity-guided isolation with an NMR-based identification led to the isolation of 4-acetylantroquinonol B (<b>1</b>), along with seven compounds. The structure of new compounds (<b>4</b> and <b>5</b>) was elucidated by spectroscopic experiments, including MS, IR, and NMR analysis. The anti-inflammatory activity of all isolated compounds was assessed at non-cytotoxic concentrations. 4-Acetylantroquinonol B (<b>1</b>) was the most potent compound against poly I:C-induced NO production in RAW 264.7 cells with an IC₅₀ value of 0.57 ± 0.06 μM.