The emergence and increasing of multidrug resistant bacteria have become an urgent thread requiring the development of a new antimicrobial class. This study was planned to investigate the effects of calcium oxide nanoparticles (CaO-Nps) on S. aureus. So, 140 samples from different sources, such as raw milk, milk by-products, intestinal contents of poultry, animal semen, and hand swabs of milkers and workers were collected. The findings illustrated S. aureus isolated from 30 samples (21.42%) using bacterial isolation which confirmed using PCR technique. Their Antibiogram pattern showed high resistance to cefoxitin, oxacillin, tetracycline, and gentamicin while were sensitive to lenzolid and clindamycin. The concentration of Cao-NPs in the prepared solution was determined by atomic absorption apparatus, and the other characters were detected by high-resolution transmission electron microscopy (HRTEM). The HRTEM showed that the size of Cao-NPs ranged from 31 to 120 nm, with a mean of 67.62 NM. The crystalized Cao-NPs show of 11.291/nm, 9.431/nm, and 6.181/diameter. The results also revealed an average zeta potential (ZP) of the prepared CaO NPs of -18.00 mV in 418 mJ. This proved that CaO NPs have a high degree of stability. Its antimicrobial activity against S. aureus was studied by a well diffusion method. The results showed that the inhibition zone diameter varied from 8 mm to 16 mm starting from Cao NPs concentration 100 NPs/ml to 600NP/ml, while 50 NPs/ml didn't cause inhibition.