Pathogenic bacterial biofilms invading surfaces in food and medical fields are a challenge to overcome. Despite all the strategies applied to fight their formation, the microbiological risk associated to bacterial biofilms remains an important threat for at risk population and for food and healthcare sectors. The prevention of biofilm formation might be an effective approach to confront this problem. In this study, stainless steel surfaces were functionalized by nisin, a natural antimicrobial peptide. The mechanism of action of immobilized nisin against sensitive bacteria is not fully understood. Therefore, nisin was grafted onto the surface by either its carboxylic group or its amino group. The generated coating's chemical, topographical and antibacterial properties were studied to understand the nisin mode of action, when immobilized, and identify the section of the bacteriocin responsible for the antimicrobial activity. The antimicrobial activity of the elaborated coatings was tested against Listeria monocytogenes. Indeed, the surfaces coated with nisin linked by its amino group showed an efficient antibacterial activity while the surface with nisin linked by its carboxylic group showed less antimicrobial effect. The antimicrobial results showed almost 2 log reduction of colony forming units for efficient antibacterial coatings while the other showed no bacterial reduction. The surface properties analysis permitted to understand the chemical and topographical characteristics of treated surfaces including nisin conformation and quantification. A tight relation was concluded between the surface topography, the nisin conformation, and the antibacterial activity of the bacteriocin-coated surfaces.