OBJECTIVE Previous studies have shown an association between alteplase for line clearance and central line–associated bloodstream infections (CLABSIs). The objective of this study was to evaluate the use of post-alteplase antibiotics as a CLABSI reduction strategy in pediatric intensive care unit (PICU) patients. METHODS This was a single center, retrospective, observational study evaluating PICU patients from January 1, 2014, through August 1, 2021, conducted at a tertiary academic PICU. Included in this study were critically ill patients who had 1 or more central venous lines (CVLs) requiring alteplase for line clearance. The primary objective was incidence of CLABSI occurrence post alteplase administration for CVL clearance, with or without targeted single-dose antibiotics (piperacillin-tazobactam or vancomycin) post alteplase. Secondary outcomes included evaluation of total alteplase administrations and risk factors associated with CLABSI occurrence. RESULTS Two hundred fifty patients were included, with 156 receiving alteplase only, 82 piperacillin-tazobactam, and 12 vancomycin, and with median ages of 2.8, 3.8, and 3.8 years, respectively. Seven CLABSIs occurred in the alteplase-only group, with 0 incidences in both the piperacillin-tazobactam (exact OR, 0.12; exact 95% CI, <0.01–0.59; p < 0.01) and vancomycin (exact OR, 1.20; exact 95% CI, 0.03–9.80; p = 1.00) groups. Patients in the piperacillin-tazobactam group achieved statistical significance for CLABSI risk factors that may benefit by decreasing CLABSI incidence (p values <0.01–0.02). CONCLUSIONS Alteplase use has been associated with CLABSIs. Providing a single dose of post-alteplase antibiotics targeting the most likely site-specific pathogens may reduce the incidence of CLABSIs.