The present study reports the presence of thymoquinone (TQ), a promising benzoquinone compound, in the lichen Dirinaria frostii (strain DF_MSCBU, NCBI-accession no. PP955960), collected from Similipal National Park, India. The extract of D. frostii was subjected to GCMS analysis and detected the presence of thymoquinone. Further, purification of the TQ fraction was carried out using HPLC against a standard thymoquinone reference. FTIR analysis identified the key functional groups. In addition, Nuclear Magnetic Resonance (¹H NMR and ¹³C NMR) spectroscopy was performed to determine the structure and composition of the molecule. The purified TQ was then used for antibacterial assays against pathogenic bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Vibrio cholerae following the MIC method showed pathogen inhibition at different incubation time, of both Gram +ve and Gram -ve bacteria. Thymoquinone showed significant bactericidal activity compared to the standard antibiotics azithromycin, streptomycin, and erythromycin in a dose-dependent manner. This study demonstrates occurrence of thymoquinone from D. frostii as a novel finding and opens new possibilities for its therapeutic applications in the future.