Most pollutants are released into the environment in the presence of other contaminants, creating complex mixtures. In life-cycle impact assessment (LCIA) methods, characterization factors (CFs) are used to obtain the potential impacts associated with each contaminant emission. Current LCIA methods do not include CFs to evaluate the potential impacts of complex organic mixtures on ecosystems. This study explores the possibility of developing new CFs for petroleum mixtures. Petroleum products are an example of mixtures whose constituents have a common toxic mode of action: the narcosis effect. Characterization factors were calculated for a series of representative constituents of a specific petroleum mixture and also for different fractions of the same mixture developed using the hydrocarbon block (HBM) and Total Petroleum Hydrocarbon Criteria Working Group (TPHWG) methods. Finally, CFs were developed for the mixture itself as a whole by using experimental property measurements and estimations. The soil-water partitioning coefficient, water solubility, degradation kinetic constant in soil, octanol-water partitioning coefficient, and vapor pressure were measured while the molar weight and the degradation kinetic constants in air, water, and sediments were estimated. The highest aquatic ecotoxicological CFs, no matter the approach chosen, were obtained for an emission to freshwater up to 2.2 × 10(+07) PAF·m(3) ·d/kg for the highest CF. CF distributions obtained using the different blocking method and experimental CFs obtained for oil as a whole are, on average, not significantly different, given the known uncertainty of ecotoxicological models in LCIA. Consequently, all the CFs obtained using the different blocking methods from the literature are considered relevant for characterizing the potential impact for aquatic ecotoxicity of petroleum substances.