Clinical guidelines recommend that children with pleural empyema be treated with chest tube insertion and intrapleural fibrinolytics. The addition of dornase alfa (DNase) has been reported to improve outcomes in adults but remains unproven in children. To determine if intrapleural tissue plasminogen activator (tPA) and DNase is more effective than tPA and placebo at reducing hospital length of stay in children with pleural empyema. This multicenter, parallel-group, placebo-controlled, superiority randomized clinical trial included children diagnosed as having pleural empyema requiring drainage aged 6 months to 18 years treated at 6 tertiary Canadian children's hospitals. A total of 379 children were assessed for eligibility; 281 were excluded and 98 were randomized. One child was excluded after randomization for not meeting the inclusion criteria. Data were collected from March 4, 2013, to December 13, 2017. Participants underwent chest tube insertion and 3 daily administrations of intrapleural tPA, 4 mg, followed by DNase, 5 mg (intervention group), or 5 mL of normal saline (placebo; control group). Participants, families, clinical staff, and members of the study team were blinded to allocation. The primary outcome was hospital length of stay from chest tube insertion to discharge. Secondary outcomes included time to meeting discharge criteria, time to chest tube removal, mean fever duration, additional pleural drainage procedures, hospital readmissions, and total health care cost. Of the 97 analyzed children with pleural empyema, 52 (54%) were male, and the mean (SD) age was 5.1 (3.6) years. A total of 49 children were randomized to tPA and DNase and 48 were randomized to tPA and placebo. Treatment with tPA and DNase was not associated with decreased hospital length of stay compared with tPA and placebo (mean [SD] length of stay, 9.0 [4.9] vs 9.1 [5.3] days; mean difference, -0.1 days; 95% CI, -2.0 to 2.1; P = .96). Similarly, no significant differences were observed for any of the secondary outcomes. Of the 14 adverse events in the tPA and DNase group, 6 (43%) were serious; of the 21 adverse events in the tPA and placebo group, 8 (38%) were serious. There were no deaths. The addition of DNase to intrapleural tPA for children with pleural empyema had no effect on hospital length of stay or other outcomes compared with tPA with placebo. Clinical practice guidelines should continue to support the use of chest tube insertion and intrapleural fibrinolytics alone as first-line treatment for pediatric empyema. ClinicalTrials.gov identifier: NCT01717742.