Our knowledge about the possible effects of inflammatory factors, oxidative stress, and coagulation factors on the venous vessel wall in patients with chronic venous insufficiency (CVI) is limited. The role of L-Arginine and endogenous dimethylarginine in endothelial dysfunction may be essential in explaining the etiopathogenesis of venous insufficiency (VI). In this study, 35 patients diagnosed with CVI were included. Venous blood samples were collected from (A) the VI-operated leg during endovenous ablation, (B) the vena saphena magna (VSM) of a healthy leg, and (C) the antecubital vein. The L-Arginine and its metabolite levels were measured using API SCIEX 3200 LC-MS/MS methods. The statistical analysis was performed using IBM SPSS 27.0 and MedCalc 15.8. In intra-group comparison, Ornithine and LNMMA values were lower in the Leg with operated CVI compared to the Upper Extremity (p < 0.05). According to CEAP classification, healthy leg serum ornithine levels were lower in CEAP-3 compared to CEAP-2 (p < 0.05). Serum LNMMA levels in the upper extremity were higher in CEAP-3 compared to CEAP-2 (p < 0.05). Intra-group Comparison by CEAP Class, L-Arginine, and Ornithine values were lower in the CEAP-3 class operated CVI leg compared to the upper extremity (p < 0.05). The literature shows decreased circulating nitric oxide levels in patients with CVI. This study found that L-arginine and Ornithine levels were lower, especially in the operated CVI group. These findings suggest that changes in L-arginine and dimethylarginine levels may provide valuable information about the pathophysiology of venous insufficiency. Biomarkers can help monitor treatment responses and assess disease progression.