The commercial PowerPlex® Fusion kit is an autosomal STR multiplex kit that has high discrimination power and is more informative in forensic, paternity, and relationship-testing cases. Key features of this multiplex system are the possibility to direct amplify FTA™ card punches as well as non-FTA cards and commonly used swabs; optimised inhibitor tolerance and high sensitivity generating full profiles from amount as little as 100 pg of human DNA. This study focused on the optimization of performance variables such as FTA™ punch sizes, reduced reaction volumes, and FTA™ purification reagent aiming to increase the analytical sensitivity, decrease the sample consumption, and cost effectiveness. LOD and LOQ values demonstrated high sensitivity of the PowerPlex® Fusion system. In addition, population databases of Brunei Malay and Chinese from the Brunei Darussalam were established, and parameters of forensic importance were calculated. Overall, the forensic parameters indicated an enhanced utility of the PowerPlex® Fusion kit for forensic evidence analysis and paternity testing in Brunei Malay and Chinese populations.