Bacterial cellulose (BC) is recognized as a wound dressing material well-suited for chronic wounds; however, it has no intrinsic antimicrobial activity. Further, the formation of bio-films can limit the effectiveness of the pre-saturation of BC with antimicrobial agents. Here, to hinder biofilm formation by P. aeruginosa, we immobilized the hydrolytic domain of PelA (a glycohydrolase involved in the synthesis of biofilm polysaccharide Pel) on the surface of BC. The immobilization of 32.35±1.05 mg PelAh per g BC membrane resulted in an eight-fold higher P. aeruginosa cell detachment from BC membrane, indicating reduced biofilm matrix stability. Further, 1D and 2D infrared spectroscopy analysis indicated systematic re-duction of polysaccharide biofilm elements, confirming the specificity of immobilized PelAh. Importantly, BC-PelAh was not cytotoxic towards L929 fibroblast cells. Thus, we conclude that PelAh can be used in BC wound dressings for safe and specific protection against biofilm formation by P. aeruginosa.