This paper aims to identify the cytotoxic and antimicrobial properties of extracts of edible lichen, Evernia prunastri. Its extracts were prepared using different solvents (hexane, chloroform, methanol, and water). The toxicity of different extracts of oak moss was screened against five cell lines while for antibacterial potential, two bacteria were used. We also chemically profiled this lichen species to link their biological potential with the identified compounds. The findings of our study revealed a positive correlation between the elevation of cell sensitivity and the increase in the treatment doses of the extract in those higher doses applied reverberated to higher cell sensitivity. Chloroform and methanol extracts from E. Prunastri were active against the tested gram-positive and gram-negative bacterial strains. The LCMS analysis of the chloroform extract of E. prunastri yielded the maximum number of compounds compared to any previous investigation employing other analytical techniques. Obtained results suggest that E. prunastri can be prospective in biotechnology as an alternative source of antimicrobial and anticancer substances.