Microorganisms and sediments in reclaimed water adhere to the inner walls of pipes or water tanks, forming biofilms that support the continuous growth of microorganisms. These biofilms provide a protective barrier, shielding bacteria from disinfectants. This study investigated the impact of biofilms on bacterial growth and reproduction in reclaimed water and the factors limiting bacterial growth in reclaimed graywater (GMR) and reclaimed mixed wastewater (MWR). The results revealed that biofilm biomass gradually increased and reached a maximum value on Days 20–25, and the biomass of organisms continued to decrease after 40 days. Biofilms serve as a source of bacteria, continuously releasing them into reclaimed water systems. The presence of biofilms reduced the biological stability of the reclaimed water, leading to water quality deterioration. The concentration of assimilable organic carbon in the reclaimed water showed a positive correlation with the heterotrophic bacterial count and Escherichia coli levels in both the reclaimed water and biofilms. The threshold value of chlorine for inhibiting biofilms in reclaimed water was no <2 mg/L. High concentrations of free chlorine delayed the growth of biofilms but did not reduce the final biomass generated by the biofilms. Carbon was the limiting factor for the biological stability of reclaimed water, while nitrogen, phosphorus, and inorganic salts were not limiting factors. Thus, minimizing the concentration of organic matter in reclaimed water can reduce the nutrient sources available for biofilm formation. This study provides support for advancements in the wastewater reuse industry.