LAB contribute to antibacterial effect against pathogens by generating antimicrobial agents, however, a sufficient cell concentration is required. Citrulline, a non-protein amino acid, provides extra energy to LAB by arginine deiminase pathway to improve cell growth. Citrulline is also a precursor of nitric oxide (NO), which plays an important role in protecting from enteric pathogens. The aims of this study were (1) to investigate the effect of adding citrulline on NO production by LAB and its antibacterial activity, (2) to investigate the antibacterial mechanisms of LAB against foodborne pathogens, and (3) to examine the stimulating effect of NO production in the intestinal epithelial cells and its anti-adhesive effect. The selected LAB were incubated with 0, 0.1, or 0.2% of citrulline in de Man, Rogosa, Sharpe (MRS) broth and in milk at 37°C for 18–20h. The antimicrobial activities against the pathogens were determined by measuring the diameter of the zones of inhibition. The NO production ability of LAB was determined by using metmyoglobin supplemented MRS plates and the bacteriocin-like inhibitory substances (BLIS) production ability of LAB was determined by eliminating the effect of acids and hydrogen peroxide. The stimulating effect of NO production by citrulline and LAB and the anti-adhesive effect were evaluated using IPEC-J2 cell line. The selected LAB when fermented with citrulline addition in MRS and fermented milk with Lactobacillus helveticus ASCC 511 significantly inhibited the tested pathogens (all P < 0.001). Milk with added citrulline when fermented with L. helveticus ASCC 511 and L. bulgaricus ASCC 756 showed the dose-dependent effect on the inhibitory activity of Shigella sonnei ATCC 25931. None of the selected LAB were capable of producing NO for converting metomyglobin to nitrosomyoglobin in MRS-Mb agar. Addition of citrulline to milk fermented with LAB might enhance the antibacterial effect of LAB against selected pathogens in vitro. The cell culture work has shown some interesting data with regard to stimulation effect of NO production in the intestinal epithelial cells and its anti-adhesive effect.