Obesity is an important risk factor for developing severe asthma. Dietary fatty acids which are increased in sera of obese individuals and after high fat meals, activate the innate immune system and induce inflammation. This study investigated whether dietary fatty acids directly cause inflammation and/or synergise with obesity-induced cytokines in primary human pulmonary fibroblasts in vitro. Fibroblasts were challenged with BSA-conjugated fatty acids (ω-6 PUFAs, ω-3 PUFAs or SFAs) with or without TNFα and release of the pro-inflammatory cytokines, IL-6 and CXCL8, was measured. We found that the ω-6 PUFA arachidonic acid (AA), but not ω-3 PUFAs or SFAs up-regulates IL-6 and CXCL8 release. Combined AA and TNFα challenge resulted in substantially greater cytokine release than either alone, demonstrating synergy. Synergistic upregulation of IL-6, but not CXCL8 was mainly mediated via COX. Inhibition of p38 MAPK reduced CXCL8 release induced by AA and TNFα alone, but not in combination. Synergistic CXCL8 release following AA and TNFα challenge, was not medicated via a single signaling pathway (MEK1, JNK, PI3K and NF-κB), nor by hyperactivation of NF-κB or p38. To investigate if these findings occur in other airway cells, effects of AA in primary human airway smooth muscle (ASM) cells and human bronchial epithelial cells were also investigated. We found pro-inflammatory effects in ASM cells, but not epithelial cells. This study suggests that diets rich in ω-6 PUFAs might promote airway inflammation via multiple pathways, including COX dependent- and independent pathways, and in an obese person may lead to more severe airway inflammation.