Resistance to antibiotics has posed a high demand for novel strategies to fight bacterial infections. Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. However, their poor solubility in water and sensitivity to degradation has limited their application. Here, we report the design of a smart, pH-responsive antimicrobial nanobiocomposite material based on the AMP nisin and 2,2,6,6-tetramethyl-1-piperidinyloxyl-oxidized nanofibrillated cellulose (TONFC). Morphological transformations of the nanoscale structure of nisin functionalized-TONFC fibrils were discovered at pH values between 5.8 and 8.0 using small-angle X-ray scattering. Complementary ζ potential measurements indicate that electrostatic attractions between the negatively charged TONFC surface and the positively charged nisin molecules are responsible for the integration of nisin. Modification of the pH level or increasing the ionic strength reduces the nisin binding capacity of TONFC. Biological evaluation studies using a bioluminescence-based reporter strain of Bacillus subtilis and a clinically relevant strain of Staphylococcus aureus indicated a significantly higher antimicrobial activity of the TONFC–nisin biocomposite compared to the pure nisin against both strains under physiological pH and ionic strength conditions. The in-depth characterization of this new class of antimicrobial biocomposite material based on nanocellulose and nisin may guide the rational design of sustainable antimicrobial materials.