Metal nanomaterials have attracted extensive attention in biological labeling and imaging due to their controllable physical and chemical properties. Recently, a lot of effort has been devoted to preparing various ultrasmall and functional copper nanoclusters (CuNCs) with different emissions from blue to red, soluble both in organic and aqueous phases. Herein, a novel one-step synthetic method is proposed for the preparation of stable water-soluble glutathione-capped (GSH-capped) CuNCs. The resulting nanoclusters have a good dispersibility and stability in aqueous media. The stability was examined by several test experiments. The mass detection in ESI-HRMS mode allowed ionization of several doubly charged species with formula Cu5L6, Cu6L6, Cu7L6, Cu8L6, and Cu9L6 (L = C10H16N3O6S). The use of advanced separation techniques including liquid chromatography (HPLC), gel electrophoresis (PAGE), and capillary electrophoresis (CE) allowed the separation of several clusters, some of which are larger than Au25(GS)18, as shown by PAGE. To the best of our knowledge, this is the first report on successful size-separation of CuNCs. Moreover, the synthesized CuNCs show a dose-dependent antimicrobial effect. At lower cluster concentration the growth of bacteria is partially reduced. However, at higher concentrations, the bacterial growth is completely restricted.