Globally, the amounts of metal ore deposits have been declining, so the research directions investigating the extraction of metals from materials that are classified as waste are gaining more importance every year. High concentrations of Cu, Pb, Zn, and Fe were analyzed in the sludge sediment (Zlaté Hory, Czech Republic), which is a waste product of the mining industry. In the bioleaching process, bacterial cells have been established as being able to convert metals from solid to liquid phase. However, the most important parameters of bioleaching are particle size, pH, and pulp density, thus our research focused on their optimization. The acidophilic and mesophilic bacteria Acidithiobacillus ferrooxidans were applied due to the high Fe content in the sample. The recovery of metals in the leachate was determined by F-AAS and the residual metal concentrations in the waste fraction were analyzed by XRF. The grain size fractions <40 µm –200 µm were investigated. The atomic absorption spectrometry (AAS) results show that the highest Fe (76.48%), Cu (82.01%), and Pb (88.90%) recoveries were obtained at particle size of 71–100 μm. Zn was dissolved for all fractions above 90%. Experiments with different pH values were performed at a pH of 1.6–2.0. The highest dissolution rates of Zn, Fe, and Cu were achieved with a suspension pH of 1.8, where 98.73% of Zn, 85.42% of Fe, and 96.44% of Cu were recovered. Due to the high percentage dissolution of metals, experiments were performed under pilot conditions in a bioreactor at a pulp density of 2.5% and 4.2% (w/v). From an economic point of view, the leaching time of 28 days was evaluated as sufficient.