Even though there is evidence of decreasing trends of per- and polyfluoroalkyl substances (PFAS) in Czechia, there are still major sources of PFAS pollution. Regarding the still-inconsistent results of the relationship between cardiometabolic health and PFAS, the present study sought to determine the association between PFAS levels and the presence of cardiometabolic biomarkers, including blood pressure and dysglycemia drivers in the Czech population. A cross-sectional study with 479 subjects (56.4% women, median: 53 years, range: 25–89) was conducted. Four PFAS were measured in serum: perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluorooctane sulfonate (PFOS). The associations between natural log (ln)-transformed PFAS and cardiometabolic biomarkers were assessed through generalized additive models using linear regression and smoothing thin plate splines, adjusted for potential confounders. There were positive and significant (p < 0.05) associations between the ln-transformed PFOA and glucose (β = 0.01), systolic (β = 0.76) and diastolic blood pressure (β = 0.65); total cholesterol (β = 0.07) and LDL-c (β = 0.04); and PFOS with glucose (β = 0.03), BMI (β = 2.26), waist circumference (β = 7.89), systolic blood pressure (β = 1.18), total cholesterol (β = 0.13), and HDL-c (β = 0.04). When significant, the correlations of PFNA and PFDA were negative. Of the four PFAS, only PFOA and PFOS showed a positive association, even in serum levels not as high as the values from the literature.