Purposes: Proposing to use the optimal antiseptics with adequate drying time as skin preparations to decrease the burden of blood culture contamination in the emergency department is common. However, other clinical characteristics and conditions might also have the effects on the blood culture contamination. Methods: An intervention cross-sectional study was conducted in an ED with approximately 86,000 annual census hospital during August 2011 to December 2012. Patients were randomly selected to receive one of the three different anti-septic agents prior to invasive procedures: 0.5% alcoholic chlorhexidine, 2% alcoholic chlorhexidine, or 10% povidone in 95% alcohol, followed with 75% alcohol. Clinical characteristics for blood culture contamination and effects of different anti-septic agents were assessed. Results: A total of 57,898 eligible ED patients were enrolled. Old age, end stage of renal disease (ESRD) patients, severe triage, and malignancy were more likely to have blood culture contamination in the ED. Patients treated with 2% alcoholic chlorhexidine group may not significant improve blood culture contamination in the ED but has the lowest contamination rate. Conclusions: Applying 2% alcoholic chlorhexidine as anti-septic might minimize the skin commensals was suitable for the high patient census EDs to improve the care quality. However, other underlying conditions might still be associated with blood contamination in the ED.