OBJECTIVE This study aimed to elucidate the benefits of nanoformulation of doxorubicin (DOX) and thymoquinone (TQ) loaded with nanofibers of poly-N-acetyl glucosamine (pGlcNAc), which is known as F2 gel, over their conventional free forms. Moreover, evaluate the role of TQ in improving chemotherapeutic effect and ameliorating nephrotoxicity of DOX. METHODS The drugs were loaded into F2 gel followed by measurement of physicochemical characterization. Next, MCF-7 and HEPG2 cells were treated with the prepared formulations and assessed for apoptosis alongside with cellular proliferation. Furthermore, we experimentally induced Heps liver carcinoma in mice and at the end of the treatment, mice were sacrificed and serum samples were used to assess nephrotoxicity markers; blood urea nitrogen (BUN) and creatinine. Additionally, renal tissue was used for determination of oxidative markers and antioxidant enzymes; whereas, tumor tissue was utilized to measure nuclear factor kappa B (NF-κB) and caspase 3. RESULTS Nanoformulation showed dramatic increase in apoptosis, caspase 3, and antioxidant enzymes; in contrast to, dramatic fall in cell viability, tumor volume, oxidative and nephrotoxicity markers, and NF-κB compared to corresponding free therapies. Combined therapy was superior in conserving the measured parameters compared to other treated groups. CONCLUSIONS F2 gel loaded with DOX and TQ revealed enhanced antitumor activity with minimal toxicity. Moreover, using TQ as an adjuvant with DOX could augment its cytotoxicity and ameliorate nephrotoxicity.