Some bacterial species form biofilms in suboptimal growth and environmental conditions. Biofilm structures allow the cells not only to optimize growth with nutrient availability but also to defend each other against external stress, such as antibiotics. Medical and bioengineering implications of biofilms have led to an increased interest in the regulation of bacterial biofilm formation. Prior research has primarily focused on mechanical and chemical approaches for stimulating and controlling biofilm formation, yet optical techniques are still largely unexplored. In this paper, we investigate the biofilm formation of Bacillus subtilis in a minimum biofilm-promoting medium (MSgg media) and explore the potential of optical trapping in regulating bacterial aggregation and biofilm development. Specifically, we determine the most advantageous stage of bacterial biofilm formation for optical manipulation and investigate the impact of optical trapping at different wavelengths on the aggregation of bacterial cells and the formation of biofilm. The investigation of optically regulated biofilm formation with optical tweezers presents innovative methodologies for the stimulation and suppression of biofilm growth through the application of lasers.