The biocidal activities of a series of quaternized polyurethanes were examined against Staphylococcus aureus and Escherichia coli. The percentage of dead cells on a surface was found to depend on the alkyl halide used for quaternization, the concentration of quaternized moieties in the polyurethane, the gram-type of the microorganism, and the contact time of the organism with the surface. N,N-bis(2-hydroxyethyl)isonicotinamide (BIN) was incorporated as the chain extender in a series of poly(tetramethylene oxide)-based polyurethane block copolymers. Three families of materials were synthesized that contained increasing hard segment fractions and therefore increasing concentrations of BIN. The pyridine ring in BIN was quaternized with a variety of alkyl halides to form cationic polyurethanes that possessed biocidal activity. The effect of quaternization on material properties was examined with tensile testing, water absorption analysis, and contact angle measurements. The antibacterial action of the polymers was investigated with zone of inhibition experiments and fluorescence microscopy, which was established as a reliable technique to determine the viability of organisms attached to a polymer surface.