Naturally occurring antioxidants present in bilberry were separated into three groups: vitamin C, phenolic acids and flavonoids using solid phase extraction (SPE). Chemical composition of bilberry extract fractions was obtained by spectrophotometry and high performance liquid chromatography (HPLC). The results of HPLC analysis point out the high content of vitamin C (1529 μg/g) in fraction Fr1, flavonoids (1328.58 μg/g) in fraction Fr2 and phenolic acids (494.31 μg/g) in fraction Fr3. The free radical scavenging activities of these antioxidant fractions on superoxide anion radicals and detection of free radical intermediates was studied using electron spin resonance (ESR) spectroscopy. Superoxide anion free radical assay demonstrated very potent free radical scavenging activity of bilberry extract fractions. The results of correlation analysis showed that the separated classes of antioxidants from bilberry (vitamin C, flavonoids and phenolic acids) are responsible for its antioxidant activity.