The associations of folate biomarkers and polycyclic aromatic hydrocarbons (PAHs) in the general population remain unclear. Therefore, this study aimed to examine whether folate biomarkers are associated with PAHs, and whether folate biomarkers can mitigate adverse health outcome caused by PAHs. This prospective cohort study included 11,246 participants from the National Health and Nutrition Examination Survey (NHANES), which documented 1303 deaths over a mean follow-up of 9.1 years. Multivariable linear regression models were used to examine the relationship between urinary individual PAHs and folate biomarkers. Multivariable Cox proportional hazards regression models were used to calculate hazard ratios and 95 % CIs for the associations of PAHs and folate biomarkers with CVDs mortality and all-cause mortality. We found negative associations between folate in red blood cells (RBC) and urinary 1-Hydroxyphenanthrene (percentage change for a 2·7 fold-increase in folate −4.19 %, 95 % CI -5.80 % to −2.56 %CI), 2-Hydroxyfluorene (−6.66 %, −7.84 % to −5.49 %), 3-Hydroxyfluorene (−5.78 %, −6.77 % to −4.78 %)) and 1-Hydroxynapthalene (−2.75 %, −3.48 % to −2.01 %). The associations between serum folate and PAHs were consistent with those observed for RBC folate, and negative associations were also found between serum folate and 2-Hydroxynapthalene (−4.10 %, −5.26 % to −2.94 %). Within the lowest quartile of folate levels in RBC, there are strong associations of 2-Hydroxyfluorene, 3-Hydroxyfluorene, 1-Hydroxynapthalene, and 2-Hydroxynapthalene with elevated risk of CVDs mortality [HRs (95 % CI) >1]. As folate levels in RBC increase to the third and fourth quartiles, these associations no longer exist [HRs (95 % CI) <1, P-interaction<0.05]. The positive associations between urinary PAHs and CVDs mortality are also eliminated as serum folate levels rise (HRs (95 % CI) <1, P-interaction<0.05). Furthermore, we also found higher levels of folate in both RBC and serum can greatly reduce the adverse impact of 1-Hydroxynapthalene on all-cause mortality. Consistent results were also validated in daily dietary folate and the folic acid supplement intake. Our study highlighted a robust negative relationship between urinary PAHs and folate. Additionally, folate was found to effectively mitigate mortality caused by PAHs, although we did not observe a direct reduction in mortality attributable to folate.