GC-FID and GC-MS were used to determine the chemical composition of the essential oil of the aerial parts of Heracleum orphanidis Boiss. Also, the HPLC-DAD/ESI-ToF-MS profile of the methanol extracts of aerial parts and roots was determined. The main components of the essential oil were n-octanol, octyl hexanoate and n-octyl acetate, while coumarins were the most prevalent compounds in methanol extracts. An evaluation of antioxidant activity showed that the methanol and aqueous extracts of the aerial parts had the highest potential. In terms of antimicrobial activity determined by microdilution assays, the essential oil and methanol extract of the roots showed the greatest effectiveness. The colonies of Pseudomonas aeruginosa PAO1 treated with H. orphanidis samples produced less toxic pyocyanin, showed lower twitching and flagella mobility and biofilm formatting was reduced. The analyses in this study showed the considerable biological potential of H. orphanidis considering free radicals and various pathogenic strains, including the wild type of P. aeruginosa.