Farsetia hamiltonii Royle is a medicinal plant of Cholistan desert, Pakistan, traditionally used for treating diabetes, oxidative stress, arthritis, fever, gastrointestinal and respiratory diseases. This study represents unprecedented phytochemical, enzymatic and biological properties of F. hamiltonii root extracts to prove floric uses. Evaluation of Phytochemical constituents was done by screening, total flavonoid, phenolic contents and gas chromatography-mass spectrometry. The phytochemical screening revealed the presence of glycosides, bounded anthraquinones, flavonoids, saponins, steroids, coumarins and diterpenes in root extracts. Eight compounds were identified in dichloromethane extract, whereas one compound was identified in methanol extract of root part of F. hamiltonii. The dichloromethane extract possesses significant lipoxygenase, chymotripsin and cholinesterase enzyme inhibition activities, whereas methanol extract possess lipoxygenase, alpha glucosidase, chymotrypsin and acetylcholinesterase enzyme inhibition activities. The antibacterial activity of methanol extract was significant against selected five microbial strains. Nine compounds were reported in root part of F. hamiltonii first time. The enzyme inhibition assays on anti-cholinesterase, anti-alpha glucosidase, antilipoxygenase, antichymotripsin and antibacterial activities were found significant for the extracts of root parts of F. hamiltonii. Therefore, results of this study justify folkloric therapeutic potential of F. hamiltonii root in treating diabetes, inflammations and infectious diseases.