Background: Knowing the local epidemiology and etiology of bloodstream infections allows tailoring the empirical initial antimicrobial therapy to obtain a better outcome for these episodes. Aim: To describe the epidemiological and microbiological aspects as well as the factors associated with mortality in patients with bloodstream infection in Colombian hospitals. Methods: Sub-analysis of a prospective cohort study of 375 consecutive patients with bloodstream infection in 10 hospitals in Colombia, admitted between September first 2007 and February 29, 2008. Results: The most frequently isolated bacteria were Gram-negative bacilli in 54% of patients, followed by Gram-positive cocci in 38.4%. The source of infection was known in 67%, unknown in 24% and associated with intravascular catheter in 9%. The most frequently isolated bacteria were Escherichia coli (46%), coagulase-negative Staphylococci (16%), Klebsiella pneumoniae (8.9%) and Staphylococcus aureus (7.8%). Staphylococcus aureus was methicillin sensitive in 82% of patients (46/56). Overall 28-day mortality was 25% and their independent associated factors were age, SOFA score and APACHE II score. Conclusions: In our study the most frequently isolated bacteria in bloodstream infections were Gram-negative bacilli, contrasting those reported in developed countries. The overall mortality rate was high and the factors associated with mortality were age and severity scores.