Gulf War Illness (GWI) affects approximately 30% of the deployed 700,000 U.S. veterans as a multi-symptomatic disease, including neurological symptoms. Here, we used Gulf War Chemical (GWC) combinations employed in preclinical GWI models to evaluate their effects in vitro on microglia. When applied directly to microglia, none of the GWC increased inflammatory cytokine secretion; GWC combinations containing corticosterone were anti-inflammatory. Pre-exposure to the GWC pyridostigmine bromide (PB) and permethrin (PM), but not other GWCs, enhanced lipopolysaccharide-induced inflammation. Similarly, only treatment of microglia with plasma from a chronic PB + PM preclinical GWI model resulted in increased inflammatory cytokines. Together, these data indicate the PB + PM preclinical GWI model combination has direct and indirect microglial priming effects, while the in vivo neuroinflammation associated with models containing DFP and corticosterone does not appear to involve humoral peripheral input or direct priming/activation of microglia.