This study aimed to enhance sludge dewatering through sequential bioleaching, employing the filamentous fungus Mucor sp. ZG-3 and the iron-oxidizing bacterium Acidithiobacillus ferrooxidans LX5. The mechanism by which Mucor sp. ZG-3 alleviates sludge dissolved organic matter (DOM) inhibition of A. ferrooxidans LX5 was investigated, and the optimal addition of energy source for enhanced sludge dewaterability during sequential bioleaching was determined. Sludge dissolved organic carbon (DOC) decreased to 272 mg/L with a 65.2% reduction by Mucor sp. ZG-3 in 3 days, and the degraded fraction of sludge DOM was mainly low-molecular-weight DOM (L-DOM) which inhibited the oxidization of Fe2+ by A. ferrooxidans LX5. By degrading significant inhibitory low-molecular-weight organic acids, Mucor sp. ZG-3 alleviated DOM inhibition of A. ferrooxidans LX5. In the sequential bioleaching process, the optimal concentration of FeSO4·7H2O for A. ferrooxidans LX5 was 4 g/L, resulting in the minimum specific resistance to filtration (SRF) of 2.60×1011 m/kg, 40.0% lower than that in the conventional bioleaching process with 10 g/L energy source. Moreover, the sequential bioleaching process increased the sludge zeta potential (from -31.8 to -9.47 mV) and median particle size (d50) of the sludge particle (from 17.90 to 27.44 μm), contributing to enhanced sludge dewaterability. Inoculation of Mucor sp. ZG-3 during the bioleaching process reduced the demand for energy sources by A. ferrooxidans LX5 while improving sludge dewaterability performance.