Most studies on autism spectrum disorder (ASD) risk factors have been conducted in developed countries where ethnicity and environment are different than in developing countries. We compared nutritional status, immune response and microbiota composition in mestizo children with ASD with matched controls in Ecuador. Twenty-five cases and 35 controls were matched by age, sex and school location. The prevalence of under-and overweight was higher in children with ASD. Nutritional differences were accompanied by abnormal food habits and more frequent gastrointestinal symptoms in children with ASD. Also, greater serum concentrations of TGF-β1 were observed in children with ASD. Finally, there was greater alpha diversity and abundance of Bacteroides (2 OTUs), Akkermansia, Coprococcus and different species of Ruminococcus in ASD children.