Carbon quantum dots (CQDs) and their derivatives have potential applications in the field of biomedical imaging. Toxicity is one of the critical parameters that can hamper their success in biological applications. In this context, our goal was to systematically investigate both in vivo and in vitro toxicity of nitrogen doped carbon quantum dots (NCQDs). In vivo toxic effects were evaluated for 30 days in Swiss albino mice at two different concentrations (5.0 mg per kg body weight (BW) and 10.0 mg per kg BW) of NCQDs. Results of haematological, serum biochemical, antioxidant and histopathological parameters showed no noteworthy defects at both of these concentrations. An in vitro assessment was performed against the human cervical cancer cell line (HeLa cells) at the concentration of 0–400 μg ml⁻¹. The LDH profile, DNA fragmentation, apoptosis, and growth cycle of cells showed no apparent toxicity of NCQDs. The overall study offers highly biocompatible N-doped carbon quantum dots, which may be considered as an attractive material for future biomedical applications.