Objectives: Lichen is a stable mutually beneficial organism. Composed of fungi, a Mycobiont is united with algae, a phycobiont or cyanobacteria. Since the ancient period, the extract derived from Lichens is utilized in the medication of numerous diseases. An attempt was made to isolate and structure elucidation of the erythrin, a biologically active compound from corticolous lichen Roccella montagnei, and analyze the antimicrobial and antioxidant activities of lichen’s acetone extract under in vitro conditions. Methods: An attempt was made to isolate and characterized the erythrin, a biologically active compound from corticolous lichen R. montagnei using ultraviolet, Fourier transform infrared, High-performance liquid chromatography, nuclear magnetic resonance, and gas chromatography–mass spectrometry techniques. The antimicrobial activities of lichen extract were evaluated against six pathogenic microorganisms using the standard disc diffusion technique. For in vitro antioxidant activity, the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) scavenging method, hydrogen peroxide scavenging method, and phosphomolybdenum assay were done. Results: The outcomes show that the lichen extract has the maximum effective antibacterial activity compared to antifungal activity against pathogenic microorganisms. The maximum zone of inhibition was recorded in Escherichia coli (8.75±0.61 mm). Among fungal pathogens, the extract inhibited the growth of Candida albicans with a maximum inhibition zone of 7.50±0.52 mm. In vitro antioxidant activity, the DPPH scavenging method, IC50 value of lichen extract, and ascorbic acid were found to be 45.70 μg mL-1 and 39.74 μg mL-1, respectively. In the hydrogen peroxide scavenging method, it was observed that the IC50 value of lichen extract, ascorbic acid, and rutin were found to be 39.39 μg mL-1, 40.66 μg mL-1, and 45.58 μg mL-1, respectively. The maximum antioxidant content of 44.66 mg/g ascorbic acid equivalents was observed in the 100 μg mL-1 lichen extract in the phosphomolybdenum assay. Total phenolic content was higher in lichen extract with 214.84±14.84 mg gallic acid equivalent (GAE/g lichen extract). Conclusion: The present study did the isolation and structure elucidation of erythrin obtained from R. montagnei from Anaikatty and reveals that the lichen extract has the potential to control the human pathogenic microorganisms in the future and the study also suggested that the lichen extract possesses active antioxidant substances to scavenge free radicals.