Rectorite (REC) was used to prepare the intercalated nanocomposites with the biopolymer, N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC). Characterization by XRD and TEM revealed that HTCC were intercalated into the intergallery of REC. Moreover, it was confirmed from FT-IR, XRD and zeta potential analyses that interaction between HTCC and REC took place. In vitro antimicrobial assays indicated that all nanocomposites exhibited strong inhibition against Gram-positive bacteria, Gram-negative bacteria and Fungi under weak acid, water and weak basic condition, particularly against Gram-positive bacteria. The lowest minimum inhibition concentration values of the nanocomposites against Staphylococcus aureus and Bacillus subtilis were less than 0.00313% (w/v) in all media tested, and the killing rate on S. aureus reached more than 90% in 30min. The mechanism of the antimicrobial action was briefly discussed, suggesting that the fine dispersion and the interaction between HTCC and REC had a strong effect on the antimicrobial activity of the nanocomposites.