The aim of this work was to determine the most relevant pollutants in Danube surface water and wastewater in the city of Novi Sad, and to conduct a risk assessment approach on substances for the optimization of future monitoring programmes. According to the requirements of Serbian law, obligatory and operating monitoring was focused on physico-chemical and biological parameters while the expanded monitoring programmes have not been applied – often due to practical circumstances. Novi Sad, with a population of more than 300.000 inhabitants, does not have a wastewater treatment plant, and about 2 m³ of wastewater is discharged directly to the Danube river every day. Screening analyses of the water within the Danube basin around city of Novi Sad included more than 300 different organic substances, while target analysis was conducted for all WFD pollutants. The methodology for generating the list of priority substances was applied for the first time in the Danubian region around the city of Novi Sad. By applying the prioritization procedure, 86 organic substances were determined in screening analyses and 27 substances obtained within target analyses were identified as the most relevant. In addition, seasonal variation analysis was conducted to determine the occurrence trends of specific compounds in different seasons.