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Abstract Microorganism contamination on substrate surfaces is arousing increasingly concern as a serious health issue. In this research work, antimicrobial water-based acrylic paint containing silver nanoparticles (Ag NPs) was prepared using the facile Ag+ in situ reduction process, in which AgNO3 and reducing agent sodium acrylate were refluxed with acrylic polymeric solution to obtain an antimicrobial and antifungal polymeric material for substrate coating. The Synthesized antimicrobial and antifungal water-based acrylic paint were characterized by different spectroscopic techniques. The FTIR and UV–Visible spectroscopic analyses were investigated to study the water-based acrylic paint structure as well as the significant impact of Ag NPs on the paint matrix. The UV–Visible and FTIR Spectra peak shows successful integration of Ag NPs within the polymer matrix without altering the core functional groups of the paint. The water based acrylic paint exhibited a strong antimicrobial activity, revealed substantial inhibition zones against all four strains of Gram negative represented by Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Gram-positive represented by Bacillus cereus. The coated film on substrate also shows great inhibition zone which exhibit a strong antimicrobial activity. Moreover, water based acrylic paint also exhibited a great antifungal activity, revealed substantial zone of inhibition against the Aspergillus niger, Aspergillus terreus and Rhizopus arrhizus fungal strains. Also, the coated film showed the best adhesion at 50% and 80% solution of polymeric coating sample as compared to pure or very dilute sample coating. This innovative approach has the potential to revolutionize varies industries from healthcare to construction.