Background & objectives:This study aims to assess the antibacterial efficacy of montmorillonite nanoclay modified with silver and bismuth nanoparticles against Escherichia coli, a common gram-negative bacterium known for causing gastrointestinal and extraintestinal infections. Methods: Methods: In this research, the initial steps for producing montmorillonite nanoclay included extracting bentonite from mines, crushing and grinding the raw material, and then sieving the resulting powder to obtain particles smaller than 74 μm. The nanoclay underwent optimization and was treated with silver and bismuth nanoparticles. The analysis involved electron microscopy, X-ray diffraction, laser diffraction for particle size assessment, and evaluation of quartz peak intensity to measure the effectiveness of the antibacterial nanoclay synthesis. The Minimum Inhibitory Concentration (MIC) was determined through agar dilution to assess its impact on E. coli viability in vitro. Results: Utilizing Results: Using montmorillonite particles smaller than 2 μm was effective in producing pharmaceutical nanoclay. The montmorillonite nanoclay with silver showed the highest efficacy against the standard E. coli strain, with a MIC value of 64 μg/mL. Following in effectiveness were the bismuth nanoparticle-modified nanoclay and the montmorillonite nanoclay, having MIC values of 128 and 512 μg/mL, respectively. Conclusion: The physical production method emerged as the safest and most efficient approach for pharmaceutical-grade nanoclay. Treating nanoclay with silver ions showed greater success compared to bismuth nanoparticle modification.