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Background: The concept of multinephron segment diuretic therapy (MSDT) has been recommended in severe diuretic resistance with only expert opinion and case-level evidence. The purpose of this study was to investigate the safety and efficacy of MSDT, combining 4 diuretic classes, in acute heart failure (AHF) complicated by diuretic resistance., Methods and Results: A retrospective analysis was conducted in patients hospitalized with AHF at a single medical center who received MSDT, including concomitant carbonic anhydrase inhibitor, loop, thiazide, and mineralocorticoid receptor antagonist diuretics. Subjects served as their own controls with efficacy evaluated as urine output and weight change before and after MSDT. Serum chemistries, renal replacement therapies, and in-hospital mortality were evaluated for safety. Patients with severe diuretic resistance before MSDT were analyzed as a subcohort. A total of 167 patients with AHF and diuretic resistance received MSDT. MSDT was associated with increased median 24-hour urine output in the first day of therapy compared with the previous day (2.16 L [0.95-4.14 L] to 3.08 L [1.74-4.86 L], P = .003) in the total cohort and in the Severe diuretic resistance cohort (0.91 L [0.43-1.43 L] to 2.08 L [1.13-3.96 L], P < .001). The median cumulative weight loss at day 7 or discharge was -7.4 kg (-15.3 to -3.4 kg) (P = .02). Neither serum sodium, chloride, potassium, bicarbonate, or creatinine changed significantly relative to baseline (P > .05 for all)., Conclusions: In an AHF cohort with diuretic resistance, MSDT was associated with increased diuresis without changes in serum chemistries or kidney function. Prospective studies of MSDT in AHF and diuretic resistance are warranted., (Copyright © 2021 Elsevier Inc. All rights reserved.)