Matching of charge periodicity between a guest and a host enabled effective immobilization of highly water-soluble antibiotic drug, gentamicin C, in a bentonite clay by cation exchange. X-ray diffraction, infrared spectroscopy and CHNS analysis revealed the immobilization manner of gentamicin C, which was immobilized between bentonite layers via periodic charge-charge interaction with tilted arrangement, as a trication. Both gentamicin alone and a gentamicin/bentonite hybrid were coated onto a polyurethane substrate using water-borne polyurethane binder. The antibiotic character of both films was investigated as prepared or after immersion in phosphate-buffered saline till 5 days against E. coli and B. subtilis bacteria. It was clearly shown that the gentamicin/bentonite hybrid-coated film showed sustained antibacterial efficacy even after exposure to phosphate-buffered saline, while gentamicin only-coated film gradually lost its performance under the same condition.