Background The U.S. National Healthcare Safety Network (NHSN) has provided a definition of mucosal barrier injury–associated, laboratory-confirmed bloodstream infection (MBI-LCBI) to improve infection surveillance. To date there is little information about its impact in pediatric oncology centers in low- to middle-income countries. Objectives To determine the impact of the definition on the rate of central line-associated bloodstream infection (CLABSI) and compare the clinical characteristics of MBI vs. non-MBI LCBI cases. Methods We retrospectively applied the NHSN definition to all CLABSIs recorded at a pediatric oncology center in Tijuana, Mexico, from January 2011 through December 2014. CLABSI events were re-classified according to the MBI-LCBI definition. Clinical characteristics and outcomes of MBI and non-MBI CLABSIs were compared. Results Of 55 CLABSI events, 44% (24/55) qualified as MBI-LCBIs; all were MBI-LCBI subcategory 1 (intestinal flora pathogens). After the number of MBI-LCBI cases was removed from the numerator, the CLABSI rate during the study period decreased from 5.72 to 3.22 infections per 1,000 central line days. Patients with MBI-LCBI were significantly younger than non-MBI-LCBI patients (P=0.029) and had a significantly greater frequency of neutropenia (100% vs. 39%, P=0.001) and chemotherapy exposure (87% vs. 58%, P=0.020) and significantly longer median hospitalization (34 vs. 23 days, P=0.008). Conclusion A substantial proportion of CLABSI events at our pediatric cancer center met the MBI-LCBI criteria. Our results support separate monitoring and reporting of MBI and non-MBI– -LCBIs in low- to middle-income countries to allow accurate detection and tracking of preventable (non-MBI) bloodstream infections.