Background: The present study reveals the synthesis of copper oxide nanoparticles (CuO NPs) by probiotic bacteria (Lactobacillus casei subsp. casei) and demonstrates the cytotoxic effects of these nanoparticles against gram negative and positive bacteria and cancer cell lines. Methods: The CuO NPs are biosynthesized from Lactobacillus casei subsp. casei (L. casei) in an eco-friendly and cost-effective process. These nanoparticles are characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffractometer (XRD), field emission scanning electron microscopy (FESEM), energy dispersive X-ray (EDX) and transmittance electron microscope (TEM) analysis. The antibacterial activity is examined by Well-diffusion, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) assays using Broth microdilution. Anticancer effects of these nanoparticles are evaluated by methyl thiazolyl diphenyl-tetrazolium bromide (MTT) assay and Griess test. Results: Our results confirm the biosynthesis of CuO NPs from L. casei. Antibacterial assays demonstrate that treatment of gram-negative and gram-positive bacteria with CuO NPs inhibits the growth of these bacteria. Furthermore, the cell viability of human cancer cells decreases while treated by nanoparticles. These nanoparticles increase nitric oxide (NO) secretion determined by NO production measurement. Conclusion: These results suggest that CuO NPs may exert antibacterial effects as well as cytotoxic effects on cancer cells by suppressing their growth, increasing the oxidative stress and inducing apoptosis.