The incidence of inflammatory bowel disease is increasing all over the world, especially in industrialized countries. The aim of the present work was to verify the anti-inflammatory activity of metabolites. In particular, cell-free supernatants of Lactobacillus acidophilus, Lactobacillus casei, Lactococcus lactis, Lactobacillus reuteri, and Saccharomyces boulardii have been investigated. Metabolites produced by these probiotics were able to downregulate the expression of PGE-2 and IL-8 in human colon epithelial HT-29 cells. Moreover, probiotic supernatants can differently modulate IL-1β, IL-6, TNF-α, and IL-10 production by human macrophages, suggesting a peculiar anti-inflammatory activity. Furthermore, supernatants showed a significant dose-dependent radical scavenging activity. This study suggests one of the mechanisms by which probiotics exert their anti-inflammatory activity affecting directly the intestinal epithelial cells and the underlying macrophages. This study provides a further evidence to support the possible use of probiotic metabolites in preventing and downregulating intestinal inflammation as adjuvant in anti-inflammatory therapy.