The present study reveals the anti-arthritic potential of traditionally used Parmotrema tinctorum (Pt) on experimental rats and purification of novel Isophthalic ester derivative. Arthritis was induced in rats using Freund's complete adjuvant (CFA) and subsequently treated with Pt extract (100 & 200 mg/kg.b.w). Assessment of antiarthritic activity was carried out using paw volume, arthritic score, haematological, biochemical, tissue antioxidant, histopathology and radiological analyses of ankle joints. The results revealed that continuous administration of Pt reduces the complication associated with arthritis by inhibiting the edema formation and arthritic score significantly (P < 0.05). The altered changes in biochemical parameters were brought back with an improvement in free radical scavenging ability after treatment with Pt significantly. Further purification of Pt using conventional column chromatography led to the isolation of four compounds and the structure of these isolated compounds were elucidated on the basis of spectral data's FT-IR, 1 H NMR, 13 C NMR, DEPT-NMR spectroscopy, COSY & HSQC-NMR spectroscopy and LC-MS. The spectral data revealed that the three compounds were found to be Methyl-γ-Orsellinate, Atranorin, and usnic acid (CI-III) along with a novel secondary metabolite, (C-IV)4-Hydroxy-5-methyl-isophthalicacid3-(3,4-dihydroxy-5-methyl-phenyl)ester (C 16 H 14 O 7 ,318.1amu). The present study reveals that Parmotrema tinctorum reduces complications associated with arthritis and the compounds were isolated for the first time., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)