HPLC isolation and spectroscopic characterization studies, along with the examination of DPPH radical scavenging capacity, were performed for identifying the major coumarin constituents of the bark extract of Aesculus hippocastanum L. collected from Turkey. The isolation process was carried out using a semi-preparative RP-HPLC with diode array detector. The major phenolic components of bark extract were identified by means of analytic RP-HPLC. Isolated compounds were characterized when necessary, along with comparison of available spectroscopic database, by UV-vis, H-1, C-1(3) NMR, FTIR, and HR-MS techniques. Radical scavenging capacity was determined by spectrophotometric DPPH assay. The isolated compounds A and B, in accordance with the RP-HPLC elution profile, were identified as 6-(beta-D-glucopyranosyloxy)-7-hydroxy-2H-1-benzopyran-2-one and 6,7-dihydroxycoumarin, whereas the third isolate compound C was characterized as 7,8-dihydroxy-6-methoxycoumarin. Compounds A and B exhibited important spectroscopic features with the emission maxima of 515nm and 445nm, respectively. The crude extract from A. hippocastanum bark revealed a remarkably high DPPH radical scavenging capacity with the EC50 value of 11.94 +/- 0.41 mu g/mL. The isolated components A, B, and C also exhibited scavenging capacities with EC50 values of 260.9 +/- 3.3, 4.83 +/- 0.04, and 494.8 +/- 16.4 (in mu g/mL), in the order of respective RP- HPLC profile.