Data concerning the influence of sequence variants of metabolizing enzymes on the effect modulation of current exposure to fumes of bitumen in humans are limited. To assess effect modulation of genetic variants of metabolizing enzymes, 18 single nucleotide polymorphisms (SNPs) in metabolic enzymes were analyzed regarding their impact on the urinary levels of metabolites of polycyclic aromatic hydrocarbons. Based on personal ambient monitoring data for bitumen emissions a group of 180 workers exposed to fumes of bitumen under high processing temperatures during a shift were divided into two exposure groups: 24 with high exposure (> 10 mg/m3) and 156 with low (≤ 10 mg/m3) exposure. Additionally, 55 roadside construction workers without exposure to bitumen but with similar working tasks served as a reference group. SNP determination was performed by real-time PCR using the LightCycler technology based on DNA aliquots from blood samples. The impact of sequence variants on the urinary levels of 1-OH pyrene (1-OHP) and sum of OH-phenanthrenes (OHPhe) was estimated with mixed linear models, adjusted for exposure, smoking, and other factors. Bitumen exposure was a significant predictor of the urinary concentrations of 1-OHP and OHPhe. Both metabolite levels were modulated by the GG variant of the cytochrome P450 SNP CYP3A4 −392A > G , which showed increased levels of 1-OHP (p = 0.013) and OHPhe (p = 0.001). The GG variant of EPHX1 15543A > G showed a significant effect (p = 0.006) only on OHPhe. Selected variants of metabolic enzymes can modulate the metabolite levels under exposure to bitumen. The SNP-related modulation of these biomarkers did not exceed a factor of two and is more pronounced for OHPhe than for 1-OHP.