Abstract Yellow gentian (Gentiana lutea L.), a medicinal plant widely used in traditional medicine, displays multiple biological effects, ranging from beneficial to toxic. Since many promising applications have been reported so far, our aim was to evaluate its potential concentration- and time- dependent cytotoxic and genotoxic effects in vitro. To that end we exposed human peripheral blood mononuclear cells to 0.5, 1, and 2 mg/mL of yellow gentian root extract (YGRE) to determine its effects on oxidative stress parameters [pro/antioxidant balance (PAB) and lipid peroxidation], DNA damage (alkaline comet assay and chromosome aberrations), and cell viability (trypan blue exclusion test). Cell viability decreased with increasing concentrations and treatment duration. Only the lowest YGRE concentration (0.5 mg/mL) increased oxidative stress but produced minor DNA damage and cytotoxicity. At higher concentrations, redox parameters returned to near control values. The percentage of chromosome aberrations and percentage of DNA in the comet tail increased with increased YGRE concentration after 48 h and declined after 72 h of treatment. This points to the activation of DNA repair mechanism (homologous recombination), evidenced by the formation of chromosomal radial figures after 72 h of treatment with the highest YGRE concentration of 2 mg/mL. Our results suggest that YGRE, despite induction of cytotoxic and genotoxic effects, activates cell repair mechanisms that counter oxidative and DNA lesions and induce cell death in highly damaged cells. Therefore, observed protective effects of yellow gentian after longer exposure could be a result of activated repair and removal of cells with irreparable damage.