The effect of chitosan oxidation with sodium periodate on the ability to form complexes with the dyes methylene blue, malachite green and acid fuchsin, which have an antiseptic effect, was investigated. A non-covalent interaction occurred for methylene blue and malachite green, while a covalent interaction occurred for acid fuchsin. Fourier-transform infrared spectroscopy confirmed the formation of chitosan composites with these dyes. The antimicrobial effect of these dyes complexed with oxidised chitosan against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Escherichia coli was investigated. The non-covalent attachment of malachite green and methylene blue to chitosan preserved their antimicrobial effect; hence, complexes of these dyes with oxidised chitosan provide a more convenient form that is less staining. However, the covalent conjugation of acid fuchsin with oxidised chitosan caused a significant loss of the antimicrobial activity. The developed method of conjugation of specific dyes with chitosan can be used to create complexes of this biopolymer with a wide range of biologically active organic substances.