Previous studies suggest that autism spectrum disorders are characterized by alterations in the microbiota–gut–brain axis. Probiotics may modify the composition and the functionality of the gut microbiota of autism spectrum disorder individuals, with possible cascading effects on brain function. In this study, we analyzed possible brain modifications induced by the administration of probiotics in 46 children with autism spectrum disorder using electroencephalography. A randomized 6-month controlled trial was performed. In subjects treated with probiotics, we observed a decrease of power in frontopolar regions in beta and gamma bands, and increased coherence in the same bands together with a shift in frontal asymmetry, which suggests a modification toward a typical brain activity. Electroencephalography measures were significantly correlated with clinical and biochemical measures. These findings support the importance of further investigations on probiotics’ benefits in autism spectrum disorder to better elucidate mechanistic links between probiotics supplementation and changes in brain activity. Lay abstract This study investigates the effects of a probiotic on preschoolers’ brain electrical activity with autism spectrum disorder. Autism is a disorder with an increasing prevalence characterized by an enormous individual, family, and social cost. Although the etiology of autism spectrum disorder is unknown, an interaction between genetic and environmental factors is implicated, converging in altered brain synaptogenesis and, therefore, connectivity. Besides deepening the knowledge on the resting brain electrical activity that characterizes this disorder, this study allows analyzing the positive central effects of a 6-month therapy with a probiotic through a randomized, double-blind placebo-controlled study and the correlations between electroencephalography activity and biochemical and clinical parameters. In subjects treated with probiotics, we observed a decrease of power in frontopolar regions in beta and gamma bands, and increased coherence in the same bands together with a shift in frontal asymmetry, which suggests a modification toward a typical brain activity. Electroencephalography measures were significantly correlated with clinical and biochemical measures. These findings support the importance of further investigations on probiotics’ benefits in autism spectrum disorder to better elucidate mechanistic links between probiotics supplementation and changes in brain activity.