Nanographene oxides (NGO) have shown exclusive potential in improving plants defense system mechanisms under various environmental conditions. This study was performed to estimate the potential impact of various NGO concentrations (150, 300, and 450 mu g mL (-1)) on the biochemical and morpho-physiological features of Lepidium sativum L. calli under control and sodium chloride-induced salinity stress conditions. Accordingly, a significant increase was observed in all of the studied biochemical traits of the salinity-treated calli (1.5% w/v NaCl). Moreover, NGO affected the callus related traits, secondary metabolites and enzymatic antioxidant activity under both non-salinity and salinity stress conditions. In this regard, at a concentration of 300 mu g mL (-1) NGO, the total phenolics content (TPC) (27%), total flavonoids content (TFD) (91%), phenylalanine ammonia lyase (PAL) activity (176%), 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity (140%) and relative callus fresh weight (12%) significantly increased as compared to the salinity stressed NGO-treatment. The highest increase of anthocyanins (Ant) (285%) was observed under the 450 mu g mL (-1) NGO treatment, as compared to the respective control under salinity stress. However, catalase (CAT) activity and callus growth rate under NGO elicitation significantly decreased as compared to the respective control under salinity stress. Our findings recommended that low to medium concentrations of NGO (150 and 300 mu g mL (-1)) could be applied as novel elicitors to alleviate the negative effects of salinity stresses by increasing phenolics biosynthesis in plants under in vitro conditions.