Abstract: The accumulation of organic and inorganic contaminants on the membrane surface, known as fouling, presents a major challenge to desalination plants by decreasing efficiency and raising operating costs. This study investigates the efficacy of three chemical cleaning protocols (A, B, and C) on reverse osmosis (RO) membrane fouling and microbial communities. Using various characterization techniques and microbial analysis, changes in membrane morphology, organic foulant composition, and microbial community were examined in fouled, virgin (unused), and cleaned membranes. Scanning electron microscopy analysis (SEM) revealed dense biofilm layers on fouled membranes, indicating decreased efficiency and potential structural damage. However, all three cleaning protocols effectively removed these biofilms, demonstrating their efficacy. Fourier Transform Infrared Spectroscopy (FTIR) analysis revealed interactions between foulants and membrane surfaces, with aggressive chemical cleaning partially removing foulants, leading to irreversible fouling. Thermogravimetric analysis (TGA) analysis supported these findings, showing no changes in membrane surface properties after cleaning. Microbial characterization revealed significant reductions in microbial counts after cleaning, with cleaning A, showing the most promising results. 16s rRNA amplicon sequencing identified dominant bacterial phyla including Proteobacteria, Firmicutes, and Actinobacteria on fouled membranes, with variations after cleaning. Cleaning A significantly reduced microbial counts and restored microbial community composition to a state resembling virgin membrane. Archaeal communities were also identified, except Cleaning A. Overall, this study enhances understanding of RO membrane cleaning protocols, emphasizing the importance of tailored cleaning strategies for efficient fouling mitigation and microbial management. Cleaning A emerges as the most effective protocol for restoring membrane performance and microbial community composition, laying the groundwork for improved water purification systems.