Emissions of motor vehicles, spilled and released oil, particles of vehicle's tires as well as substances from asphalt road surfaces represent important source of diffuse pollution in urban environment. During rainfall, these pollutants with various hazardous impacts could be washed off from roads into aquatic environment. The purpose of our work was to evaluate the possibility of application of biological activator made of enzymes, bacteria and nutrients in order to activate and improve the biodegradation of oils, grease and hydrocarbons in highways stormwater runoff basins. Several laboratory experiments were performed and biodegradation of pollution was monitored by chemical oxygen demand (COD), Dissolved Organic Carbon (DOC), biochemical oxygen demand (BOD5) and specific analysis. Biological activators were introduced as single or multiple additions. It has been confirmed that application of activators increased degradation of organic pollution (up to 60%), but it should be added only once in a low concentration. The application of activator was necessary only in the case of heavier pollution (rain event after a long dry season), while highway stormwater runoffs usually degraded enough during retention in collection basins. If biological activator was added to a low polluted highway stormwater runoff, its addition could even lead to the increase in the summary pollution.