Few studies have assessed the time to blood culture positivity as a predictor of clinical outcome in bloodstream infections (BSIs). The purpose of this study was to evaluate the time to positivity (TTP) of blood cultures in patients with Staphylococcus aureus BSIs and to assess its impact on clinical outcome. We performed a historical cohort study with 91 adult patients with S. aureus BSIs. TTP was defined as the time between the start of incubation and the time that the automated alert signal indicating growth in the culture bottle sounded. Patients with BSIs and TTPs of culture of </=12 h (n = 44) and >12 h (n = 47) were compared. Septic shock occurred in 13.6% of patients with TTPs of </=12 h and in 8.5% of patients with TTP of >12 h (P = 0.51). A central venous catheter source was more common with a BSI TTP of </=12 h (P = 0.010). Univariate analysis revealed that a Charlson score of >/=3, the failure of at least one organ (respiratory, cardiovascular, renal, hematologic, or hepatic), infection with methicillin-resistant S. aureus, and TTPs of </=12 h were associated with death. Age, gender, an APACHE II score of >/=20 at BSI onset, inadequate empirical antibiotic therapy, hospital-acquired bacteremia, and endocarditis were not associated with mortality. Multivariate analysis revealed that independent predictors of hospital mortality were a Charlson score of >/=3 (odds ratio [OR], 14.4; 95% confidence interval [CI], 2.24 to 92.55), infection with methicillin-resistant S. aureus (OR, 9.3; 95% CI, 1.45 to 59.23), and TTPs of </=12 h (OR, 6.9; 95% CI, 1.07 to 44.66). In this historical cohort study of BSIs due to S. aureus, a TTP of </=12 h was a predictor of the clinical outcome.