A novel approach was investigated for the assessment of leaching from a one-component polyurethane (1C-PU) coating used for hydraulic structures using nontarget analysis via LC-QTOF-MS. Leaching behavior of the 1C-PU coating was studied using experiments in which the coating was exposed to water for defined periods (6 h, 24 h, 3 d, 14 d). Three hardening durations for the 1C-PU coating were tested (0 h, 24 h, 14 d) as well as two water matrices (ultrapure water and river water), including a successive water renewal. Dissolved organic carbon, total nitrogen bound and nontarget measurements via LC-QTOF-MS showed that under all tested conditions organic substances were leached out of the 1C-PU coating. The shorter the hardening duration and the longer the leaching duration, the higher were the number and quantities of the eluted substances, while the influence of the water matrix was minor with respect to substance elution. Based on the MS2 spectra from the LC-QTOF-MS measurements, 30 substances released from the 1C-PU coating were tentatively identified. These substances belong to five chemical groups: derivatives of (i) N-(tosyl)carbamate, (ii) p-toluenesulfonamide (PTSA), (iii) 4,4'-methylenediphenyl diisocyanate (4,4'-MDI), (iv) toluenediisocyanate (TDI), and (v) oligo(ethylene) ([C2H4O]n) as well as p-toluenesulfonic acid. The identity of seven substances was confirmed by authentic reference standards, all of which exhibited an elevated bacterial toxicity to Aliivibrio fischeri. p-Toluenesulfonic acid was present in a German canal (Teltowkanal) with concentrations of up to 11 μg L-1.