Although a growing number of studies have found a relationship between delayed appropriate antibiotic therapy and mortality, few have attempted to quantify the temporal association between delayed appropriate antibiotic therapy and mortality. This study was designed to measure the elapsed time associated with an increased risk of 30-day mortality among patients with Pseudomonas aeruginosa bacteremia. The retrospective cohort study was conducted among immunocompetent, adult patients with P. aeruginosa bacteremia onset at least 2 days after hospital admission between 1 January 2001 and 30 September 2006. Classification and regression tree analysis (CART) was used to identify the delay in appropriate antibiotic therapy that was associated with an increased risk of 30-day mortality. During the study period, 100 patients met the inclusion criteria. The CART-derived breakpoint between early and delayed treatment was 52 h. The delayed treatment group experienced a >2-fold significant increase in 30-day mortality compared to the early treatment group (44 and 19%, respectively, P = 0.008). Delayed appropriate therapy of >52 h (odds ratio [OR] = 4.1; 95% confidence interval [CI] 1.2 to 13.9, P = 0.03) was independently associated with 30-day mortality in the multivariate analysis. Antibiotic resistance > or =3 classes (adjusted OR [AOR] = 4.6; 95% CI = 1.9 to 11.2, P = 0.001) and chronic obstructive pulmonary disease (AOR = 5.4; 95% CI = 1.5 to 19.7, P = 0.01) were independently associated with delayed appropriate therapy of >52 h. The data strongly suggest that delaying appropriate therapy for approximately 2 days significantly increases the risk of 30-day mortality in patients with P. aeruginosa bloodstream infections.