We aimed to assess the appropriateness of routine blood culture testing parameters and antimicrobial therapy for patients with suspected bloodstream infection. We conducted a cross-sectional study of blood cultures registered in the microbiological laboratory at a university-affiliated hospital from 4 to 15 June 2007. Using a structured implicit chart review, two infectious disease specialists assessed the appropriateness of the testing parameters and antimicrobial therapy. Overall, 2,696 blood culture bottles were collected from 260 patients during their stay, including 955 bottles that were evaluated during the study period. The indication of fungal and bacterial blood cultures was rated as appropriate for 75% (95% confidence interval [CI], 65-83) and 91% (95% CI, 87-95) of patients. Only 45% (95% CI, 39-52) of patients had an adequate number of blood cultures (i.e., two to four). An optimal volume of blood (i.e., ≥10 mL) was inoculated in 13% (95% CI, 11-15) of adult bottles. Empirical antimicrobial therapy was appropriate for 60% (95% CI, 43-76) of patients with positive blood cultures. There is room for improvement regarding routine blood culture testing parameters and antimicrobial therapy. The effectiveness of multifaceted interventions in altering the appropriateness of blood culture parameters deserves further research.