The biocompatibility and very high specific area of cellulose nanofibrils (CNF) are properties of high interest for the development of active substrates for new medical device development. Enzyme pretreated CNF (CNF-e) can be self-organized into nanostructured membranes that are suitable for active principle ingredients (API) encapsulation through adsorption phenomena. In addition, tunable surface chemistry of CNF-e, allow for covalent immobilization of API. In this work, ciprofloxacin is integrated to CNF-e membranes according to two different strategies. The first one relies only on adsorption mechanisms; ciprofloxacin is encapsulated in the bulk before the membrane formation by solvent casting. The influence of the membrane properties and preparation parameters such as grammage, thickness and drying technique, are assessed with water uptake measurements and API release experiments. The second strategy deals with the covalent immobilization of ciprofloxacin directly onto CNF-e membrane. The two kinds of membranes are then compared in terms of antibacterial activity, in both static and dynamic conditions. Thick CNF-e membranes loaded with adsorbed ciprofloxacin that were overdried (2 h, 150 °C) prove to be more resistant in liquid medium and present a more prolonged drug release. However, these membranes rapidly lost their antibacterial activity, while CNF-e membranes with covalently immobilized ciprofloxacin remain contact active for several days. These 100% CNF active nanostructured membranes can be used as new wound dressing for topical application.