Introduction: Selligueain A derived from the roots of Polypodium feei was shown to have anti-inflammatory activity, which was tested in vivo on the rats’ paw edema induced by carrageenan. The aim of this study was to evaluate the anti-inflammatory mechanism of selligueain A in vitro against the production of pro-inflammatory mediators. Methods: In this study, RAW264.7 cells were used as an inflammatory cell model, and observations were made on the inflammatory mediators nitric oxide (NO), inducible nitric oxide synthase (iNOS), and tumour necrosis factor-α (TNF-α). The NO concentration was measured by the Griess reaction, and the iNOS enzyme and the TNF-α concentrations were determined by the ELISA method. Cell viability was assessed by the [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) test. Results: Selligueain A at concentrations of 100 and 150 µM suppressed the production of NO, iNOS, and TNF-α in RAW264.7 cells stimulated by lipopolysaccharide (LPS). The concentration of 150 µM showed the highest inhibition of NO, iNOS, and TNF-α mediators with the percentage inhibition of 64.85, 55.01, and 48.54%, respectively. Conclusion: This study shows that selligueain A has anti-inflammatory activity through inhibition of NO, iNOS, and TNF-α production in RAW264.7 macrophage cells.