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A new approach to diabetic foot infections (DFIs) has been investigated, using a nisin-biogel combining the antimicrobial peptide (AMP) nisin with the natural polysaccharide guar-gum. Since in in vivo conditions bacteria may be exposed to decreased antimicrobial concentrations, known as subinhibitory concentrations (sub-MICs), effects of nisin-biogel sub-MIC values corresponding to 1/2, 1/4 and 1/8 of nisin’s minimum inhibitory concentration (MIC) on virulence expression by six <i>Staphylococcus aureus</i> DFI isolates was evaluated by determining bacteria growth rate; expression of genes encoding for staphylococcal protein A (<i>spA</i>), coagulase (<i>coa</i>), clumping factor A (<i>clfA</i>), autolysin (<i>atl</i>), intracellular adhesin A (<i>icaA</i>), intracellular adhesin D (<i>icaD</i>), and the accessory gene regulator I (<i>agrI</i>); biofilm formation; Coa production; and SpA release. Nisin-biogel sub-MICs decreased bacterial growth in a strain- and dose-dependent manner, decreased <i>agrI</i>, <i>atl</i> and <i>clfA</i> expression, and increased <i>spA</i>, <i>coa</i>, <i>icaA</i> and <i>icaD</i> expression. Biofilm formation increased in the presence of nisin-biogel at 1/4 and 1/8 MIC, whereas 1/2 MIC had no effect. Finally, nisin-biogel at sub-MICs did not affect coagulase production, but decreased SpA production in a dose-dependent manner. Results highlight the importance of optimizing nisin-biogel doses before proceeding to in vivo trials, to reduce the risk of virulence factor’s up-regulation due to the presence of inappropriate antimicrobial concentrations.