Plastic is a material commonly used in everyday objects, and its improper disposal and accumulation have become a serious environmental concern. Microplastics (MP) are small particles of synthetic organic polymers measuring less than 5 mm and pose a significant threat to the environment due to their persistence and inherent toxicity. The biodegradation of polymers depends on various factors, such as the physical and chemical properties of the polymer, environmental conditions, and the presence of microorganisms. Although biodegradation is often used as a process to remove MP from the environment, its effectiveness relies on optimizing significant process factors. The aim of this research was to investigate the biodegradation of polystyrene (PS) particles using the bacterium Bacillus subtilis. The objective was to identify significant factors influencing the biodegradation process of PS microplastics (MP) using Bacillus subtilis and to optimize the biodegradation conditions using this bacterial culture. The experiment was designed using a full factorial design (FFD) to study the effects of three factors, namely MP size, rotational speed, and optical density (OD) of the bacterial suspension, at three levels. The biodegradation process was monitored for 30 days, during which the total colony-forming units (CFU) of bacteria and the values of total carbon (TC), organic carbon (TOC), and inorganic carbon (TIC) were measured. LC/MS analysis was conducted to detect the release of additives from PS MP and the formation of potentially toxic degradation products. Changes in the structure of PS were examined using ATR-FTIR spectroscopy. The ecotoxicity of the filtrate was assessed using the bacterium Vibrio fischeri. A statistical analysis of variance (ANOVA) was used to evaluate the experimental results.