Microplastics (MP) are a global environmental problem because they persist in the environment for long periods of time and negatively impact aquatic organisms. Possible solutions for removing MP from the environment include biological processes such as bioremediation, which uses microorganisms to remove contaminants. This study investigated the biodegradation of polystyrene (PS) by two bacteria, Bacillus cereus and Pseudomonas alcaligenes, isolated from environmental samples in which MPs particles were present. First, determining significant factors affecting the biodegradation of MP-PS was conducted using the Taguchi design. Then, according to preliminary experiments, the optimal conditions for biodegradation were determined by a full factorial design (main experiments). The RSM methodology was applied, and statistical analysis of the obtained models was performed to analyze the influence of the studied factors. The most important factors for MP-PS biodegradation by Bacillus cereus were agitation speed, concentration, and size of PS, while agitation speed, size of PS, and optical density influenced the process by Pseudomonas alcaligenes. However, the optimal conditions for biodegradation of MP-PS by Bacillus cereus were achieved at γMP = 66.20, MP size = 413.29, and agitation speed = 100.45. The best conditions for MP-PS biodegradation by Pseudomonas alcaligenes were 161.08, 334.73, and 0.35, as agitation speed, MP size, and OD, respectively. In order to get a better insight into the process, the following analyzes were carried out. Changes in CFU, TOC, and TIC concentrations were observed during the biodegradation process. The increase in TOC values was explained by the detection of released additives from PS particles by LC-MS analysis. At the end of the process, the toxicity of the filtrate was determined, and the surface area of the particles was characterized by FTIR-ATR spectroscopy. Ecotoxicity results showed that the filtrate was toxic, indicating the presence of decomposition by-products. In both FTIR spectra, a characteristic weak peak at 1715 cm-1 was detected, indicating the formation of carbonyl groups (-C=O), confirming that a biodegradation process had taken place.