The staphylococcal nuclease, encoded by the nuc1 gene, is an important virulence factor of Staphylococcus aureus. However, the physiological role of the nuclease has not been fully characterized. The current study observed that biofilm development could be prevented in staphylococcal nuclease-producing strains of S. aureus; however, when the nuc1 gene was knocked out, the ability to form a biofilm significantly increased. Scanning electron and confocal scanning laser microscopy were used to evaluate the role of the nuc1 gene in biofilm formation. Moreover, the nuc1 gene product, staphylococcal nuclease, and recombinant NUC1 protein were found to have a visible effect on other biofilm-forming bacteria, such as Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae, and Haemophilus parasuis. The current study showed a direct relationship between staphylococcal nuclease production and the prevention of biofilm development. The findings from this study underscore the important role of staphylococcal nuclease activity to prevent biofilm formation in S. aureus. They also provided evidence for the biological role of staphylococcal nucleases in other organisms.