Constructing the novel and multifunctional (magnetic and anti-bacterial) nano-support for immobilization of enzymes with anti-biofilm properties stays immensely important. As both the treatment targets, dispersing the biofilms and killing the bacteria could be achieved. Therefore, in this study, we fabricated a multifunctional nano-carrier from halloysite nanotubes (HNTs) modified with Fe3O4 NPs and 3-aminopropyltriethoxysilane (AS), designed for ‘immobilization of anti-biofilm enzyme protease type I (PtI) (HNTs-Fe3O4-AS-PtI)’ and ‘anti-bacterial effect.’ The HNTs-Fe3O4-AS showed excellent PtI immobilization with 87 % of the activity recovery and 59.56 mg/g loading capacity, respectively. The materials were characterized by XRD, TGA, FT-IR, XPS, VSM, SEM, and HR-TEM analysis. Further, HNTs-Fe3O4-AS-PtI was applied for anti-biofilm activities against multiple bacterial biofilms (E. coli, S. aureus, and MRSA). The obtained results from CV staining, SEM image analysis, and live and dead assay using CLSM corroborated HNTs-Fe3O4-AS-PtI as potential anti-biofilm material against all studied bacterial biofilms. Further, HNTs-Fe3O4-AS-PtI were found to be anti-bacterial in nature. Hence, along with anti-biofilm activity, HNTs-Fe3O4-AS-PtI decipher the anti-bacterial effect. Thus, this study provided a highly effective anti-biofilm nano-formulation with anti-bacterial potential. This advanced material can be applicable in many fields, where the treatment of bacterial biofilms is critical.