The extensive use of antibiotics over the last decades is responsible for the emergence of multidrug-resistant (MDR) microorganisms that are challenging health care systems worldwide. The use of alternative antimicrobial materials could mitigate the selection of new MDR strains by reducing antibiotic overuse. This paper describes the design of enzyme-based antimicrobial cellulose beads containing a covalently coupled glucose oxidase from Aspergillus niger (GOx) able to release antimicrobial concentrations of hydrogen peroxide (H2O2) (≈ 1.8 mM). The material preparation was optimized to obtain the best performance in terms of mechanical resistance, shelf life, and H2O2 production. As a proof of concept, agar inhibition halo assays (Kirby-Bauer test) against model pathogens were performed. The two most relevant factors affecting the bead functionalization process were the degree of oxidation and the pH used for the enzyme binding process. Slightly acidic conditions during the functionalization process (pH 6) showed the best results for the GOx/cellulose system. The functionalized beads inhibited the growth of all the microorganisms assayed, confirming the release of sufficient antimicrobial levels of H2O2. The maximum inhibition efficiency was exhibited toward Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli), although significant inhibitory effects toward methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus were also observed. These enzyme-functionalized cellulose beads represent an inexpensive, sustainable, and biocompatible antimicrobial material with potential use in many applications, including the manufacturing of biomedical products and additives for food preservation.