We designed and fabricated highly efficient and selective antibacterial substrates, i.e. surface non-cytotoxic against mammalian cells but exhibiting strong antibacterial activity. For that purpose, microporous substrates (pore sizes in the range of 3-5 μm) were fabricated using the Breath Figures approach (BFs). These substrates have additionally a defined chemical composition in the pore cavity (herein either a poly(acrylic acid) or the antimicrobial peptide Nisin) while the composition of the rest of the surface is identical to the polymer matrix. As a result, considering the differences in size of bacteria (1-4 μm) in comparison to mammalian cells (above 10 µm) the bacteria were able to enter in contact with the inner part of the pores where the antimicrobial functionality has been placed. On the opposite, mammalian cells remain in contact with the top surface thus preventing cytotoxic effects and enhancing the biocompatibility of the substrates. The resulting antimicrobial surfaces were exposed to Staphylococcus aureus as a model bacteria and murine endothelial C166-GFP cells. Superior antibacterial performance while maintaining an excellent biocompatibility was obtained by those surfaces prepared using PAA while no evidence of significant antibacterial activity was observed at those surfaces prepared using Nisin.