Chlorocresol nanoemulsion disinfectant (CND) is an environmental disinfectant prepared with nanoemulsion as its drug carrier. This study aimed to investigate the bactericidal effect of CND on Staphylococcus aureus (S. aureus) and its effect on bacterial ultrastructure. The neutralizing effect of CND against S. aureus was first screened by suspension quantitative evaluation experiment procedure of neutralizer. Disinfection performance was evaluated by the determination of Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), quantitative bactericidal experiment, and comparative experiment of disinfection performance between 0.1% CND and 0.1% chlorocresol aqueous solution. Meanwhile, the effect of CND on the ultrastructure of S. aureus was investigated with scanning electron microscope (SEM) and transmission electron microscope (TEM) to preliminarily explore the bactericidal mechanism. The results showed that 3% Tween-80 in PBS could be screened as the neutralizer of CND against S. aureus. MIC and MBC were 100 μg/mL and 200 μg/mL, respectively. The bactericidal rates were all 100% when 0.06% and 0.08% disinfectant acted for 15 and 5 min, respectively. Furthermore, compared with 0.1% chlorocresol aqueous solution, the bactericidal effect of 0.1% CND was significantly enhanced (p<0.01). After treatment with CND for 10 min, SEM observation showed that the morphology of S. aureus cells were changed and the integrity destroyed. TEM observation showed that the cell shape changed, and the structures of the cell wall, cell membrane and cytoplasm were damaged in varying degrees. CND showed the strong bactericidal effect on S. aureus and could cause ultrastructure alterations of S. aureus.