Biocidal coatings are known to minimize or terminate development of bacterial and fungicidal infections. In this paper, biocidal activity of seven cationic (co)polymers with amino groups—polyethyleneimine, polyallylamine, polydiallyldimethylammonium chloride/polyhexamethylene guanidine copolymer, diallyldimethylammonium chloride/SO2 copolymer, linear and hyperbranched epichlorohydrin/dimethylamine copolymers, polydiallyldimethylammonium chloride—were tested toward Gram-positive and Gram-negative cells. The polymers showed a significant biocidal effect in both aqueous solution and after formation of polymer films on the hydrophilic glass plates. Polymer films were almost completely removed by water during 10 wash-off cycles, that finally resulted in the ultrathin monolayers with a thickness of several nanometers. A polyethyleneimine film showed the most resistance to water with a 50% loss after three wash-off cycles and 75% loss after six wash-off cycles. Binding and subsequent deactivation of pathogenic microorganisms occurs on the outer surface of cationic polymer films. It is expected that a gradual polymer wash-off will allow renewal of the outer film surface and thereby restore the biocidal properties of the polycationic coatings, including those with a nanoscale thickness.