The essential oil of the air dried aerial parts of Carduus pycnocephalus L. F. Asteraceae was prepared by hydrodistillation. GC/MS analysis revealed the presence of nineteen components representing 100% of the total oil. Hexadecanoic acid (39.62%) was the prominent component of the oil. GC/MS analysis of the unsaponifiable matter of the petroleum ether extract of the aerial parts of the plant revealed the presence of nineteen compounds; sixteen compounds were identified constituting 72.80% of total unsaponifiable matter. olean-12-en-3-α-ol (20.39%), ursa- 9(11), 12-dien-3-ol (17.74%) and hexadecanoic acid (17.62%) were the major components. GC/MS analysis of fatty acid methyl ester showed the presence of twenty fatty acids methyl esters (91.38%). The main known components were 1,2benzendicarboxylic acid dimethylester (31.08), palmitic acid methyl ester (20.08%) and azelaic acid dimethyl ester (7.60%). The antimicrobial, antispasmodic and anti-inflammatory effects were studied. The volatile oil and petroleum ether extract showed no antimicrobial activity, while the petroleum ether extract showed antispasmodic, anti-inflammatory effects. This is the first study on chemical composition and biological activities of the volatile oil and lipid contents of Carduus pycnocephalus L. endemic for Saudi Arabia.