The adhesion and proliferation of bacteria on various artificial surfaces affects the functionality of these specific interfaces. To overcome the problems caused by bacterial growth on these surfaces, various antibacterial coatings were developed. In this review, we summarized most of the antibacterial surfaces prepared by the Layer-by-Layer (LbL) assembly approach and classified these LbL films based on their antibacterial mechanisms. In the first group, the bactericidal LbL assemblies which incorporate various biocides including heavy metals, antibiotics, cationic molecules, antimicrobial peptides and enzymes are able to kill surrounding or contacted bacteria. In the second group, we focused on the physical aspects of film surfaces. Bacterial adhesion resistant LbL films have been fabricated to adjust the substrate surface properties such as surface free energy (or wettability), roughness, and surface charge which may affect the adhesion of bacteria. Furthermore, as an enhancement in the antibacterial efficiency, multifunctional LbL assemblies combining both bactericidal and adhesion resistant functionalities were discussed. The advantages and limitations of these antibacterial LbL assemblies were summarized and subsequently directions for future development were proposed.