•Patients with CVD had lower activity of PON1 Lactonase (LACase) than control patients.•LACase activity might be a new potential biomarker in CVD.•LACase activity is not influenced by the genetic polymorphisms of PON1. Paraoxonase 1 (PON1) is important in the development of atherosclerosis, and it has become the subject of intensive research. Our aim was to evaluate the association of serum PON1 activity and polymorphisms with cardiovascular disease (CVD) using four different substrates. Activity of PON1-related to arylesterase (AREase and 4-CMPAse), paraoxonase (PONase), and lactonase (LACase), and polymorphisms (A-162G, T-108C, L55M, and Q192R) were evaluated in subjects with CVD, cardiovascular risk factor (CFR), and controls. An ordered logistic-regression analysis of PON1 phenotypes was performed in the CVD group with respect to the control group. Logistic-regression analysis showed that CC-108 genotype was associated with CRF and CVD. The CVD group had the lowest activities of PON1. The LACase might be a better biomarker for CVD (OR, 0.52; 95% CI, 0.44–0.61) followed by CMPAse (OR, 0.82; 95% CI, 0.77–0.86), AREase (OR, 0.98; 95% CI, 0.97–0.99) and PONase (OR, 0.99, 95% CI, 0.99–0.99). Logistic regression of PON1 phenotypes by haplotypes showed that LACase activity was not influenced by the polymorphisms and that it could be a new potential biomarker in the development of CVD. Larger scale longitudinal studies are required.