Nanocomposites of Ag with organic montmorillonite (Ag-OMMT), Ag with montmorillonite (Ag-MMT), and organic montmorillonite (OMMT) were successfully prepared via a one-step solution-intercalated method. Sodium MMT, silver nitrate, and dimethyl octadecyl hydroxy ethyl ammonium nitrate were used as precursors. X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, and energy dispersive spectroscopy analyses confirmed that the MMT layers were intercalated, and Ag + was partly reduced to silver nanoparticles with diameters within 10-20 nm in Ag-OMMT. The decomposition temperature of the organic cations in OMMT and Ag-OMMT increased to 220 ∘ C, as revealed by differential scanning calorimetrythermogravimetric analysis. The antimicrobial activity of the nanocomposites was tested by measuring the minimum inhibitory concentration (MIC) and killing rate. The MICs of Ag-OMMT against Staphylococcus aureus, Escherichia coli, and Candida albicans were 0.313, 2.5, and 0.625 mg/mL, respectively. Because of the presence of quaternary ammonium nitrate, Ag-OMMT has a better MIC against Gram-positive bacteria compared to Gram-negative bacteria and fungi. OMMT did not show antimicrobial activity against Escherichia coli and Candida albicans. In 2 h, 0.0125 mg/mL Ag-OMMT could kill 100% of S. aureus, E. coli, and C. albicans in solution, and Ag-MMT could kill 99.995% of S. aureus, 90.15% of E. coli, and 93.68% of C. albicans. These antimicrobial functional nanocomposites have the potential for application in the area of surface decoration films.