In the current paper, NiO-NPs were synthesized through a sol–gel method by the usage of a biocompatible polymer known as Arabic gum (GA) as a stabilizing agent. The structural, optical, and magnetic properties of the obtained product was investigated by the application of TGA/DTA, FT-IR, UV-Vis, XRD, FESEM/EDX, and VSM analyzes. The FESEM images of NiO-NPs displayed its spherical shape and average size of about 59 nm. The antimicrobial activity of NiO-NPs was investigated against Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis and Gram-negative bacteria including Escherichia coli, Pseudomonas aeruginosa. The achieved findings indicated that all of the bacterial resistance was attributed to NiO-NPs, except for the case of Bacillus subtilis bacteria that did not grow at the concentration of 10 mM. We also evaluated the photocatalytic activity of nanoparticles in regards to the MB degradation and observed a photocatalytic degradation efficiency of about 80% throughout the results. The cytotoxicity of NiO-NPs has evaluated towards the cancer U87MG cell and normal Nervous Neurospheres (NCs) cells lines by means of MTT method. Detailed in vitro biological activities revealed the significant therapeutic potential of NiO-NPs. This product was able to exhibit potent cytotoxicity against U87MG cancer cells (IC50: 37.84 μg mL−1).