Diabetes mellitus (DM) patients frequently develop diabetic foot ulcers (DFU) which are generally infected by a community of microorganisms, mainly Staphylococcus aureus and Pseudomonas aeruginosa. These bacteria exhibit a multi-drug resistance profile and biofilm-forming ability which represent a hurdle in the treatment of diabetic foot infections (DFI). We aimed to evaluate the potential of Nisin Z, an antimicrobial peptide (AMP), as an alternative treatment for severe DFI. Nisin Z shows antibacterial activity against Gram-positive and Gram-negative bacteria and an increased antibacterial effect against Gram-negatives when added to EDTA. As such, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Minimum Biofilm Inhibitory Concentration (MBIC), and Minimum Biofilm Eradication Concentration (MBEC) were determined for Nisin Z, Nisin Z + EDTA (0.4%), and Nisin Z + EDTA incorporated into guar gum, in order to test its efficacy against S. aureus and P. aeruginosa isolated from the same DFU. Results showed that Nisin Z added to the chelation agent EDTA displayed higher antibacterial and bacteriostatic efficacy against mono and dual co-cultures of S. aureus and P. aeruginosa, and higher antibiofilm efficiency against monocultures. Nisin Z was moderately cytotoxic at 200 µg/mL. Prospect in vivo studies are needed to confirm the potential of Nisin Z supplemented with EDTA to be used as a complement to conventional antibiotic therapy for severe DFI.