Pyranose 2-oxidase (P2Ox) has several proposed biotechnological applications such as a bio-component in biofuel cells or for carbohydrate transformations. To improve some of the catalytic properties of P2Ox from Trametes multicolor, we selected a semi-rational approach of enzyme engineering, saturation mutagenesis of active-site residues and subsequent screening of mutant libraries for improved activity. One of the active-site mutants with improved catalytic characteristics identified was V546C, which showed catalytic constants increased by up to 5.7-fold for both the sugar substrates (D-glucose and D-galactose) and alternative electron acceptors (1,4-benzoquinone, BQ and ferricenium ion, Fc(+)], albeit at the expense of increased Michaelis constants. By combining V546C with other amino acid replacements, we obtained P2Ox variants that are of interest for biofuel cell applications due to their increased k(cat) for both BQ and Fc(+), e.g., V546C/E542K showed 4.4- and 17-fold increased k(cat) for BQ compared to the wild-type enzyme when D-glucose and D-galactose, respectively, were the saturating substrates, while V546C/T169G showed approx. 40- and 50-fold higher k(cat) for BQ and Fc(+), respectively, with D-galactose in excess. This latter variant also shows significantly modulated sugar substrate selectivity, due to an increase in k(cat)/K(M) for D-galactose and a decrease in k(cat)/K(M) for D-glucose when oxygen is the electron acceptor, as well as improved catalytic efficiencies for d-galactose, regardless of the electron acceptor used. While the wild-type enzyme strongly prefers D-glucose over D-galactose as its substrate, V546C/T169G converts both sugars equally well as was shown by the kinetic constants determined as well as by biotransformation experiments.