Background and objective: Intrapleural tissue plasminogen activator/deoxyribonuclease (tPA/DNase) therapy is increasingly used in pleural infection. Bleeding risks and costs associated with tPA remain the clinical concerns. Our dose de-escalation series aims to establish the lowest effective dosing regimen for tPA/DNase. This study assesses the intrapleural use of 2.5 mg tPA/5 mg DNase for pleural infection. Methods: Consecutive patients with pleural infection treated with a starting regime of 2.5 mg tPA/5 mg DNase were included from two centres in Australia and UK. Escalation of tPA dose was permitted if clinical response was inadequate. Results: Sixty-nine patients (mean age 61.0 years) received intrapleural 2.5 mg tPA/5 mg DNase. Most (88.4%) were treated successfully and discharged from hospital without surgery by 90 days. Patients received a median of 5 [interquartile range [IQR] = 3–6] doses of tPA/DNase. Total amount of tPA used per patient was 12.5 mg [median, IQR = 7.5–15.0]. Seventeen patients required dose escalation of tPA; most (n = 12) for attempted drainage of distant non-communicating locule(s). Treatment success was corroborated by clearance of pleural opacities on radiographs (from median 27.0% [IQR = 17.1–44.5] to 11.0% [IQR = 6.4–23.3] of hemithorax, p < 0.0001), increased pleural fluid drainage (1.98 L [median, IQR = 1.38–2.68] over 72 h following commencement of tPA/DNase) and reduction of serum C-reactive protein level (by 45.0% [IQR = 39.3–77.0] from baseline at day 5, p < 0.0001). Two patients required surgery. Six patients with significant comorbidities (e.g., advanced cancer) had ongoing infection when palliated and died. Two patients experienced self-limiting pleural bleeding and received blood transfusion. Conclusion: A starting intrapleural regime of 2.5 mg tPA/5 mg DNase, with up-titration if needed, can be effective and deserves further exploration.