Although Metschnikowia pulcherrima shows promise as a biocontrol agent, its upstream development remains limited. We maximized the viable cell count of M. pulcherrima strain APC 1.2 by employing a Central Composite Design (CCD) to optimize pH and temperature. Preliminary screening identified sucrose at 20 g/L and 1600 mg YAN/L yeast extract as optimal for viable cell count (CFU/mL), with further increases in YAN providing no additional benefit. The highest viable cell count was achieved at milder temperatures (18–22°C), where the strain grew robustly across a wide pH range (3.59–6.41), while higher temperatures increased sensitivity to acidic pH and reduced viable cell count. The strain was confirmed to be Crabtree-negative, showing no ethanol production during aerobic growth on glucose. The proposed workflow enabled reproducible optimization of cultivation parameters, providing a scalable framework for industrial applications, and laying the groundwork for future studies on non-conventional yeasts in biocontrol and industrial biotechnology.