Bimetallic nanoclusters (NCs) containing gold and silver were prepared by template-assisted synthesis using human serum albumin (HSA) via a newly optimized fabrication route at 25 °C. Additionally, following this procedure, we also reproducibly synthesized further Au/Ag NCs, containing a similar Au:Ag ratio, using bovine serum albumin (BSA), lysozyme (LYZ), transferrin (Tf), and gamma-globulin (γG). The aim was to highlight the importance of experimental conditions of the synthesis (e.g., metal ion: protein molar ratio and metal and protein concentrations, as well as synthesis time, temperature, and pH) for the composition, structure, and optical features of the protein-stabilized ultra-small-sized products. Circular dichroism (CD) spectroscopy revealed that the partial unfolding of the stabilizing proteins is primarily caused by the alkaline synthesis environment rather than the nanocluster formation itself. Furthermore, X-ray photoelectron spectroscopy (XPS) and inductively coupled plasma mass spectrometry (ICP-MS) successfully confirmed the presence of mainly metallic (Au⁰) core structures alongside Ag⁰/Ag⁺ species, providing the actual metal-to-protein ratios after purification. As a new result, cytotoxicity of these bimetallic NCs was determined by using doxorubicin-sensitive Colo205 and CCD-19Lu human normal fibroblast cell lines, and their antibacterial activity was also evaluated using four different Gram-positive and Gram-negative bacterial strains.