Thousands of different kinds of lichen metabolites are being examined for their biological activities, including anticancer properties. In this context, the present study aims to assess the anti-inflammatory activity of the acetone extract of Parmotrema austrosinense mycobiont. A lipid peroxidation assay was performed with the acetone extracts of P. austrosinense mycobiont, which was further used to evaluate its anti-inflammatory efficacy using a zebrafish model. Furthermore, the histopathological study was also carried out with muscle tissues and amplification of its inflammation marker. The results revealed that the lichen compound (i.e., lecanoric acid) in the acetone extract of P. austrosinense possesses anti-inflammatory activity. Histopathology studies confirmed the decreased numbers of neutrophil cells in the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced zebrafishes, as confirmed by changes in the fishes’ weight before and after the sample treatment, prompted by TNBS inflammation. The present results also demonstrated a dose-dependent decrease in the lipid peroxidation (LPO) levels in the muscle tissues of zebrafishes. Gene amplification studies suggested that the lichen compound might perform dose-dependent downregulation of the inflammatory gene marker of the tumor necrosis factor (TNF)-α gene; this further confirms that the extract should possess anti-inflammatory activity. As per the literature, this study is one of the most complete, comprehensive in vivo anti-inflammatory analyses in which inflammation was induced in zebrafish by using 2,4,6-trinitrobenzene sulfonic acid (TNBS). Particularly, this study successfully identified a bioactive compound isolated from the lichen P. austrosinense, and which exhibited decent anti-inflammatory activity. Keywords: lichen; anti-inflammatory activity; lipid peroxidation assay; zebrafish.