This study provides an in-depth analysis of fluid inclusions (FIs) and stable isotopes from the Manondo-Choma gold prospect in southern Malawi to understand the ore-forming mechanisms, genesis, and evolution of the hydrothermal fluids responsible for gold mineralization at the Manondo-Choma gold prospect. FIs and microthermometry studies were carried out on mineralized quartz veins from the area. The Manondo-Choma gold prospect is located in the southern Malawi within the Kirk range. Gold is mostly found in quartz veins within metamorphic rocks, such as gneiss and schists, and is structurally controlled by the NE–SW ductile shear zones. Gold mineralization is linked to quartz sulfide veins. Mineralization occurs in the following three stages: early, middle, and late, of which the middle stage is more plorific. The following three FI types were recognized in the quartz veins: pure carbonic, aqueous carbonic (CO2–H2O) and aqueous (H2O–NaCl) inclusions. Gold and associated mineralization were likely precipitated due to the lowering of pressure and fluid immiscibility. Oxygen isotope data indicate that the source of ore-forming fluids at the Manondo-Choma gold prospect was largely metamorphic in origin with minor magmatic input.