<p>The large-scale use of nickel (Ni [II]) in many industrial processes results in the production of Ni-containing wastes, posing a hazardous impact on public health and the environment. The current study highlights <i>Aspergillus flavus</i>’s potential for environmental bioremediation and the impact of Ni stress on the growth kinetics, removal efficiency and bioaccumulation. The results showed that the biomass of <i>A. flavus</i> decreased at all tested Ni (II) concentrations as compared to the control. Additionally, the minimal inhibitory concentration (MIC) of <i>A. flavus</i> was 4500 mg L^− 1 on solid medium. The Ni (II) removal rate of <i>A. flavus</i> increased with increasing Ni (II) levels and peaked at 63.76% at 750 mg L^− 1, while the maximum bioaccumulation capacity (Q) reached 83.07 mg g^− 1 at 1750 mg L^− 1. Moreover, Ni (II) stress caused an increase in malondialdehyde (MDA) (137.74% at 1750 mg L^− 1). Antioxidant enzyme activities and secondary metabolites significantly increased to counteract oxidative stress before declining at extreme concentrations. Also, Fourier-transform infrared spectroscopy (FTIR) demonstrated that amide, phosphate, and sulfur groups participate in Ni (II) biosorption as accumulation sites. Furthermore, energy-dispersive X-ray (EDX) spectroscopy confirmed the presence of Ni (II) on the mycelial surface, with a measured weight% of 9.44%. Notably, our study reveals a synergistic network of defense mechanisms that support <i>A. flavus’s</i> resilience. These results emphasize the work’s novelty and substantiate the isolate’s use as a feasible option for long-term bioremediation.</p>