Water quality standards for copper are usually stated in total element concentrations. It is known, however, that a major part of the copper can be bound in complexes that are biologically not available. Natural organic matter, such as humic and fulvic acids, are strong complexing agents that may affect the bioavailable copper (Cu2+) concentration. The aim of this study was to quantify the relation between the concentration of dissolved natural organic matter and free Cu2+ in surface waters, and the biological effect, as measured in a standardized ecotoxicological test (48 h‐median effective concentration EC50 Daphnia magna, mobility). Six typical Dutch surface waters and an artificial water, ranging from 0.1 to 22 mg/L dissolved organic carbon (DOC), were collected and analyzed quarterly. Chemical speciation modeling was used as supporting evidence to assess bioavailability. The results show clear evidence of a linear relation between the concentration of dissolved organic carbon (in milligrams DOC/L) and the ecotoxicological effect (as effect concentration, EC50, expressed as micrograms Cu/L): 48‐h EC50 (Daphnia, mobility) = 17.2 × DOC + 30.2 (r2 = 0.80, n = 22). Except for a brook with atypical water quality characteristics, no differences were observed among water type or season. When ultraviolet (UV)‐absorption (380 nm) was used to characterize the dissolved organic carbon, a linear correlation was found as well. The importance of the free copper concentration was demonstrated by speciation calculations: In humic‐rich waters the free Cu2+ concentration was estimated at ≈10−11 M, whereas in medium to low dissolved organic carbon waters the Cu2+ was ≈10−10 M. Speciation calculations performed for copper concentrations at the effective concentration level (where the biological effect is considered the same) resulted in very similar free copper concentrations (≈10−8 M Cu) in these surface waters with different characteristics. These observations consistently show that the presence of organic matter decreases the bioavailability, uptake, and ecotoxicity of copper in the aquatic environment. It demonstrates that the DOC content must be included in site‐specific environmental risk assessment for trace metals (at least for copper). It is the quantification of the effects described that allows policy makers to review the criteria for copper in surface waters.