Chromium (Cr) is classified as a toxic metal as it exerts harmful effects on plants and human life. Bacterial-assisted nano-phytoremediation is an emerging and environment friendly technique that can be used for the detoxification of such pollutants. In current study, pot experiment was conducted in which spinach plants were grown in soil containing chromium (0, 5, 10, 20 mgkg⁻¹) and treated with selected strain of Bacillus sp. and Cu–O nanoparticle (CuONPs). Data related to plant's growth, physiological parameters, and biochemical tests was collected and analyzed using an appropriate statistical test. It was observed that under chromium stress, all plant's growth parameters were significantly enhanced in response to co-application of CuONPs and Bacillus sp. Similarly, higher levels of catalase, superoxide dismutase, malondialdehyde, and hydrogen peroxide were also observed. However, contents of anthocyanin, carotenoid, total chlorophyll, chlorophyll a & b, were lowered under chromium stress, which were raised in response to the combined application of CuONPs and Bacillus sp. Moreover, this co-application has significant positive effect on total soluble protein, free amino acid, and total phenolics. From this study, it was evident that combined application of Bacillus sp. and CuONP alleviated metal-induced toxicity in spinach plants. The findings from current study may provide new insights for agronomic research for the utilization of bacterial-assisted nano-phytoremediation of contaminated sites