Biological metal leaching is a technology used in the mining and biohydrometallurgy industries where microorganisms mediate the dissolution of metals and semi-metals from mineral ores and concentrates. The technology also has great potential for various types of metal-rich waste. In this study, bioleaching was used for sludge from water treatment. In addition to checking the applicability of the process to such a substrate, the influence of experimental conditions on the effectiveness of bioleaching of metals with sludge from water treatment was also determined, including sample acidification, addition of elemental sulfur, incubation temperature, and Acidithiobacillus thiooxidans-isolated strain. The measurement of metal concentration and, on this basis, the determination of bioleaching efficiency, as well as pH and oxygen redox potential (ORP), was carried out during the experiment at the following time intervals: 3, 6, 9, 12 days. After the experiment was completed, a mass balance was also prepared. After the experiment, high efficiency of the process was obtained for the tested substrate. The effectiveness of the process for most metals was high (Ca 96.8%, Cr 92.6%, Cu 80.6%, Fe 95.6%, Mg 91%, Mn 99.5%, Ni 89.7%, Pb 99.5%, Zn 93%). Only lower values were obtained for Al (58.6%) and Cd (68.4%).