As a result, even if nisin helped a greater number of antibiotic molecules to access the bacteria, the antibiotics were not able to generate sufficient antibacterial activity. In the evaluation of the antibacterial activity of the combination of penicillin and nisin, the results of the combination did not correspond with the mechanism of intracellular delivery by cell membrane disruption; however, the antibacterial activity of penicillin was still significantly strengthened in the presence of nisin. Penicillin is considered to bind to DD-transpeptidase, a penicillin-binding protein that catalyzes the last step of peptidoglycan biosynthesis and thus prevents complete cell wall synthesis. The mechanism of the cell wall disruption differs from that of nisin by Lipid II, an intermediate in the cell wall synthesis pathway. Therefore, the two antimicrobials attack the pathway differently, and are able to generate a greater disruption within the cell wall. This was also demonstrated by the TEM images. A majority of the cells were severely damaged by the dual attacks of penicillin and nisin and lost their original cell wall integrity. Similarly, this combined mechanism is seen in combinations of nisin and the cephalosporins. However, E. faecalis cells appear more resistant to the dual action of Masitinib vancomycin and nisin, and E. faecalis will still survive the challenge of the two antimicrobials in combination. Vancomycin decreases the accessibility of Lipid II by blocking the cell wall biosynthesis, and inhibites the membrane leakage activity of nisin against intact cells. Superior antibacterial activity is achieved by combining antimicrobials with different antibacterial mechanisms compared with a combination of antimicrobials with the same or similar mechanisms. Bacterial biofilms generally become 10–1,000 times more resistant to the effects of antimicrobial agents than planktonic cells. A majority of E. faecalis in the biofilm survived the challenges of penicillin, ciprofloxacin, and chloramphenicol, but nisin significantly improved the antibiofilm activities of the three antibiotics, with action taking place throughout many layers of the biofilm. Compared with some other bacteria, for example, Streptococcus mutans, E. faecalis form a biofilm that includes a substantial amount of eDNA but a low level of extracellular polysaccharides, leading to a low resistance to penetration by antimicrobial agents. Antimicrobial molecules can easily enter this biofilm, and the high antibacterial activity of the antibiotics may play a key role in the inhibition of E. faecalis biofilms. Therefore, the potent antibacterial activity resulting from the combination of penicillin and nisin resulted in superior antibiofilm characteristics against E. faecalis. However, a CLSM image collected after treatment with penicillin and nisin includes some minor green areas.