Erythrosine and tartrazine are one of the synthetic azo dye mostly consumed in food, drugs and other industrial compounds. This study was designed to investigate the adverse effect of combine erythrosine and tartrazine on cognitive and neurobehavioral functions, pro-oxidants, endogenous antioxidants, cholinergic system and pro-inflammatory cytokines in rats. Erythrosine and tartrazine (2 mg/kg, 6 mg/kg, and 10 mg/kg, b.w., p.o, 50:50) was administered to rats (n = 6) for 6 weeks. Memory and neurobehavioral assessment using Novel object recognition test (NORT) and Elevated plus maze (EPM) and biochemical (pro-oxidants and anti-oxidant enzymes) and pro-inflammatory cytokine measurement from the brain sub regions namely, hippocampus and prefrontal cortex were done at the end of treatment. The results showed (p < 0.05) significant decreased memory and neurobehavioral function, increased acetyl-cholinesterase and pro-oxidants activity (Malonaldehyde level and Nitrite), decreased endogenous anti-oxidants (Glutathione and Catalase) and increased pro-inflammatory cytokines (Tumor necrosis factor-alpha, TNF-α). We suggested that the mechanism by which this oxidative and neuro-inflammatory damage and cholinergic system alteration occur might be related to the release of metabolite in fission of the azo dyes of the combined erythrosine and tartrazine administration in the animals. However, we concluded on these findings that erythrosine and tartrazine dyes significantly provoke the release of oxido-nitrergic and neuroinflammatory stress markers and also may incite acetyl-cholinesterase activities in different brain regions leading to memory and neurobehavioral impairment.