Staphylococcus aureus is one of the most common and distributed pathogens. S. aureus was resistant to 11 antibiotics including four categories based on their functions. Synthesis of copper nanoparticles (CuNPs) from CuSO4.5H2O using ascorbic acid was performed. Properties of CuNPs using UV and TEM were measured. Results of characterization observed absorption peak at 592 nm and CuNPs cubic in shape with average size 3.5 nm. The activity of CuNPs as bactericidal agent against S. aureus was determined using agar well diffusion method. CuNPs showed greater antibacterial activity and MIC was 40 μg/ml. The anti-biofilm activity of CuNPs was assayed in 96-well polystyrene plates and 3 ml polyethylene tube. Obviously, remarkable progressive inhibition of biofilm formation was noticed with increasing of CuNPs concentrations. Determination of CuNPs cytotoxicity on WI-38 cells using MTT protocol was performed. The toxicity observed at concentration 62.5 was 23% with IC50 value 134.65 μg ml−1.