Surface sediment samples were collected in the Banja Luka city area, near heavy traffic roads and the Vrbas river basin, in order to determine the origin of the organic substance. For this purpose, soluble organic substances were extracted by the Soxhlet method, and extracts were fractionated by the method of column chromatography into a group of saturated hydrocarbons, aromatic hydrocarbons, and polar compounds. The total amount of soluble organic substance was in the range of 1185-3050 μg/kg. Fractions were analyzed by gas chromatography-mass spectrometry method using a non-polar HP-5 ms stationary phase with a He 99.999% flow rate of 1 ml/min. Based on the chromatograms characteristic for n-alkanes m/z 71, steranes m/z 217, and terpenes m/z 191, the specific n-alkanes range was determined (n-C14-n-C39). n-Alkanes domination of odd/even C-number and carbon preference index values ranged from 1.12-2.48. The calculated parameters showed that the organic substance in the samples is of sedimentary origin with a significant anthropogenic input. In addition, based on the analysis of aromatic fractions and the detected unresolved complex mixture, the microbiological degradation of organic substance was determined.