The intensive use of copper (Cu) compounds as an alternative biocide in antifouling paints (APs) has resulted in wide Cu contamination into the marine environment, especially near marina harbor activities. In this work, the applicability of Cu isotopes to discriminate Cu origins related to the use of Cu-based APs in marine environments was tested. To this, Cu isotopes in APs, shipyard sludges, and sediment cores sampled in the Cu-contaminated Mediterranean marina of Port Camargue were determined. APs represent an important dominant anthropogenic source for metals in this site, making it ideal to test Cu isotopes as tracers. The overall isotope composition of four sediment cores and a surface sample varied between −0.13 and 0.44 ‰ (δ65Cu relative to NIST-976). Selected APs brands show a similar Cu concentration ~0.15 % and δ65Cu average of 0.54 ± 0.05 ‰. The plot of δ65Cu vs concentration for all datasets allowed dissociating natural and APs end-members. However, sample isotope systematics were not consistent with a conservative mixing binary source process. Heavily Cu-contaminated sediments show isotope signatures lighter than APs brands. However, the most Cu-contaminated sample, located directly above the careening area, shows a δ65Cu slightly lighter than APs (0.44 ‰ vs 0.54 ‰, respectively). Results suggest the preferential releasing of a heavy isotope pool by APs when these compounds are solubilized in seawater. The isotope fractionation was attributed to potential chemical Cu coordination changes during its elemental partition between paint and marina seawater and the fractionation induced by the organic ligands in the water column, before deposition. Further laboratory experiments are recommended to model the isotope fractionation mechanisms related to Cu release by APs. Because the APs' isotope signature is modified in marine environments, the use of Cu isotopes as tracers of AP in marine environments is challenging and needs more investigation.