Objective The uranium pollution risk and resource value associated with stone coal waste rock stockpiles constitute a core contradiction in mine environmental management and resource recovery. Microbial leaching is a key technology for recovering low-grade uranium resources, yet the potential of mixotrophic bacteria, which combine the advantages of both autotrophic and heterotrophic metabolism, remains unclear in this field. This study investigated the uranium leaching effect and mechanism of the indigenous mixotrophic bacterium Alicyclobacillus ferrooxydans S1-24WXX from stone coal waste rock, aiming to achieve the synergy between pollution control and resource utilization. Methods The indigenous acidophilic mixotrophic strain A. ferrooxydans S1-24WXX was isolated from a stone coal mine in Shangrao, Jiangxi. Leaching experiments were conducted with three groups of organic matter addition (T_OM), no organic matter addition (T_NOM), and a sterile control (CK) to systematically evaluate the uranium leaching efficiency of this strain from uranium-rich stone coal waste rock. X-ray diffraction was used for mineral characterization of the raw ore and leaching residues. Dynamic monitoring was performed on changes in pH, redox potential (Eh), iron ion concentration, and uranium leaching rate during the process. Results Inoculation with S1-24WXX significantly enhanced the acidity (pH<2.0), Eh (≈600 mV), and Fe³⁺ concentration (≈1 000 mg/L) of the leaching system, all of which far exceeded those of the chemical leaching control group. Under T_OM and T_NOM conditions, the maximum uranium leaching rates reached 46.9% and 44.2%, respectively, which were 4.07 and 3.84 times that of the control group. Mineral analysis indicated that the strain catalyzed pyrite oxidation, leading to the formation of a strongly acidic, highly oxidizing, and Fe³⁺-rich leaching environment, which was the dominant mechanism of promoting uranium release. Conclusion This study reveals the significant potential of the mixotrophic bacterium A. ferrooxydans in uranium bioleaching. The bacterium drives uranium dissolution through inorganic acidification rather than organic acid complexation, providing a new pathway for the green recovery of low-grade uranium resources. This holds important academic value for mine environmental management and sustainable resource utilization.