The aim of this study was to assess free fatty acids' (FAs) anti-/proinflammatory capabilities and their influence on inflammatory gene expression and H O production by human peripheral blood mononuclear cells (PBMCs). Anthropometric and clinical measurements were performed in 26 participants with metabolic syndrome. Isolated PBMCs were incubated for 2 h with several free fatty acids-palmitic, oleic, α-linolenic, γ-linolenic, arachidonic and docosahexaenoic at 50 μM, and lipopolysaccharide (LPS) alone or in combination. H O production and IL6, NFκB, TLR2, TNFα, and COX-2 gene expressions were determined. Palmitic, γ-linolenic, and arachidonic acids showed minor effects on inflammatory gene expression, whereas oleic, α-linolenic, and docosahexaenoic acids reduced proinflammatory gene expression in LPS-stimulated PBMCs. Arachidonic and α-linolenic acids treatment enhanced LPS-stimulated H O production by PBMCs, while palmitic, oleic, γ-linolenic, and docosahexaenoic acids did not exert significant effects. Oleic, α-linolenic, and docosahexaenoic acids induced anti-inflammatory responses in PBMCs. Arachidonic and α-linolenic acids enhanced the oxidative status of LPS-stimulated PBMCs. In conclusion, PBMC assays are useful to assess the anti-/proinflammatory and redox-modulatory effects of fatty acids or other food bioactive compounds.