In this work, Cu-doped ZnSe nanoparticles (NPs) at the presence of different Cu contents and also with/without 6 min microwave irradiation were fabricated in aqueous medium, and then some optical properties and also their antibacterial properties against two gram-positive bacteria of Staphylococcus aureus and Bacillus cereus were investigated, employing disc-diffusion method. To fabricate these NPs, Se ion source was provided from the interaction between Se and NaBH4, and zinc acetate was used as Zn ion source. At the fixed pH of 11.2, thioglycolic acid was used as surfactant to prevent agglomeration of NPs. Previously reported results of X-ray diffraction characterization and UV-visible spectroscopy of solutions containing nanoparticles, show the range of 1.94–2.14 for particles size and 3.50–3.65 eV for energy gap. In this research, non-linear optical susceptibility of ZnSe and ZnSe:Cu nanoparticles have been determined; results imply that these nanoparticles have a high potential in optical and optoelectronic applications and among them, sample owing 1.5% of impurity and so owing the highest v(3), is an optimum candidate in optical applications. Results of the present research confirm that nonlinear optical susceptibility is in inverse relation with the energy gap and increase with increasing of Cu%. To explore the antibacterial activity of the present samples, first Staphylococcus aureus and Bacillus cereus bacteria were inoculated on Muller–Hinton–Agar culture, and then the loaded discs by nanoparticles were placed on them. After 18 h from the incubation, the inhibition zone diameters (their antibacterial sensitivity) were measured for each bacteria. Results of this research imply on that these nanoparticles have considerable antibacterial activity against the gram-positive bacteria of Staphylococcus aureus and Bacillus cereus, and have the usage ability in the field of antibacterial drugs. During this assessment, increase in impurity content resulted in improv