Multilayer films from hydrophobically modified poly(acrylic acid) (HMPA) and their complexes with cationic surfactants were successfully prepared using the layer-by-layer (LbL) method. Alkyl trimethylammonium bromide derivatives with various lengths of the hydrophobic chain (C10–C18) were used to interact with the HMPA polymer, generating highly hydrophobic domains in the films and contributing to the antimicrobial properties of the prepared coating. The antimicrobial efficiency against common pathogens such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans was investigated in relationship with the morphology and composition of the thin films. The wettability and roughness of the multilayered systems were evaluated using atomic force microscopy (AFM) and contact angle measurements. The effects of the microbial exposure on the surface properties of the prepared films were investigated in order to assess the stability of the HMPA-deposited multilayers and the durability of the antimicrobial activity. The hydrophobically modified films exhibited antimicrobial activity against the studied pathogens. The best efficiency was registered in the case of S. aureus, which showed an inhibition of growth up to 100% after 2 h. C. albicans proved to be less sensitive to the effect of the multilayers deposited from HMPA–surfactant complexes. These results suggest that HMPA and HMPA–surfactant complex LbL multilayer films can be used as promising materials in antimicrobial surface coatings with increased resistance to pathogens during exposure.