The present study was undertaken to assess the antimicrobial activity of Lactobacillus spp. (L. salivarius, L. johnsonii, L. reuteri, L. crispatus, and L. gasseri) against Campylobacter jejuni as well as their immunomodulatory capabilities. The results demonstrated that lactobacilli exhibit differential antagonistic effects against C. jejuni and vary in their ability to elicit innate responses in chicken macrophages. All lactobacilli exerted inhibitory effects on C. jejuni growth, abrogated the production of the quorum sensing molecule autoinducer-2 (AI-2) by C. jejuni and inhibited the invasion of C. jejuni in human intestinal epithelial cells. Additionally, all lactobacilli, except L. reuteri, significantly reduced the expression of virulence-related genes in C. jejuni, including genes responsible for motility (flaA, flaB, and flhA), invasion (ciaB), and AI-2 production (luxS). All lactobacilli enhanced C. jejuni phagocytosis by macrophages and increased the expression of interferon (IFN)-γ, interleukin (IL)-1β, IL-12p40, IL-10, and chemokine (CXCLi2) in macrophages. Furthermore, L. salivarius, L. reuteri, L. crispatus, and a mixture of all lactobacilli significantly increased expression of the co-stimulatory molecules CD40, CD80, and CD86 in macrophages. In conclusion, these findings demonstrate that lactobacilli possess anti-Campylobacter and immunomodulatory activities. Further studies are needed to assess their protective efficacy against intestinal colonization by C. jejuni in broiler chickens.