Aim: Drug-induced gingival overgrowth has a multifactorial nature and the pathogenesis is still uncertain. It has been suggested that Nitric Oxide (NO) might play a role in the pathogenesis of drug-induced gingival overgrowth due to the contribution of NO to immune response and matrix degradation. NO levels in biological fluids have been used as a diagnostic biomarker in many diseases. The aim of this study is to determine whether NO levels in plasma, saliva, and gingival crevicular fluid (GCF) can serve as a potential biomarker for the evaluation of drug-induced gingival overgrowth risk. Materials and Methods: A total of 104 patients, receiving cyclosporine A (n = 35), phenytoin (n = 25), nifedipine (n = 26), or diltiazem (n = 18) participated in the study. The amount of gingival overgrowth was evaluated with two indices and was given as percentage. Periodontal clinical parameters including plaque index (PI), gingival index (GI), gingival bleeding time index (GBTI), and probing depth (PD) were also assessed. Saliva, GCF, and plasma samples were obtained from each participants. Nitrite and nitrate levels in saliva, GCF, and plasma were analyzed by Griess reagent. Results: Salivary nitrite and nitrate levels in responders were significantly higher than those in non-responders in only phenytoin group (p < 0.05). Nitrite and nitrate levels of gingival crevicular fluid and plasma did not significantly differ between responders and non-responders in all study groups (p > 0.05). Salivary nitrite levels exhibited a significant correlation with PD, GBTI, severity of gingival overgrowth (%GO), and GCF volume (p < 0.05). Additionally, a strong positive correlation was detected between saliva and plasma nitrate levels (p < 0.005). However, both nitrite and nitrate levels in GCF and plasma demonstrated no significant correlation with clinical parameters, GO severity, and GCF volume (p > 0.05). Conclusion: Salivary nitrite and nitrate levels could be used as periodontal disease biomarkers in phenytoin induced gingival overgrowth, and that saliva seems to have a better diagnostic potential than GCF and plasma for the evaluation of drug-induced gingival overgrowth risk. However, when all drug groups were considered, saliva nitrite and nitrate levels could not be used as a biomarker for drug-induced gingival overgrowth.