Probiotic supplements consumed adequately at the proper time can affect health by modulating inflammatory pathways in gastrointestinal epithelial cells and modifying the resultant inflammatory response. The current study applied in vitro models to investigate the effectiveness of probiotics in modulating inflammatory pathways and altering inflammatory gene expression in gastrointestinal epithelial cells, with the ultimate goal of promoting probiotic consumption as a therapeutic and preventive measure for chronic inflammatory bowel conditions. HT-29 cells were treated with Gram-negative bacteria to evaluate the changes in pathways related to inflammation activities before and after treatment with aLactobacillusspp. cocktail (L. plantarum,L. rhamnosus,L. brevis, andL. ruteri) and aBifidobacteriumspp. cocktail (B. bifidum,B. langum, andB. breve) using the real-time PCR method and ELISA for IL-1β and IL-6 as pro-inflammatory cytokines. The results showed that the expression of NF-κB signaling pathway genes and IL-1β and IL-6 cytokines increased after exposure to Gram-negative components. In contrast, all probiotic combinations significantly decreased the expression of genes and the secretion of cytokines. However, this decrease was significantly smaller in cells that underwent probiotic treatment after inflammation induction. In addition, cocktails containing combinedLactobacillusandBifidobacteriumdemonstrated robust anti-inflammatory activity relative to solo cocktails. Our observations confirm that probiotic consumption could positively impact inflammatory conditions and alleviate inflammatory symptoms; they can be particularly effective as a preventive measure. Our study provides preliminary evidence to support the lifetime consumption of probiotics.