Introduction <p>Autism spectrum disorder (ASD) affects millions of children worldwide, negatively impacting the daily functioning and quality of life of patients and their families. New therapeutic approaches for ASD are being investigated. Probiotics have shown promise in modulating gut microbiota and ameliorating ASD symptoms. We aim to evaluate the impact of a 12-week multispecies probiotic supplementation on the severity of core autism symptoms in children with ASD.</p> Methods and analysis <p>This study is a randomized, double-blind, placebo-controlled clinical trial. One hundred and 10 children aged 7 to 15 years diagnosed with ASD will be randomly assigned in a 1:1 ratio to receive either a probiotic mixture or a placebo for 12 weeks. The probiotic mixture contains 5 × 10⁹ colony-forming units per dose of <i>Bacillus subtilis</i> W20,<i> Bifidobacterium infantis</i> W17, <i>Bifidobacterium lactis</i> W51, <i>Lactobacillus acidophilus</i> W37, <i>Levilactobacillus brevis</i> W63, <i>Lacticaseibacillus rhamnosus</i> W140<i>, Lactococcus lactis</i> W19, and <i>Propionibacterium freudenreichii</i> W200. The primary outcome will be the assessment of core autism symptoms using the Autism Symptom Rating Scales (ASRS). The secondary outcomes will include the quality of life, evaluations of sleep impairments, parental stress levels, gastrointestinal symptoms, and urinary p-cresol levels. These outcomes will be assessed twice: at baseline and after 12 weeks of intervention.</p> Ethics and dissemination <p>The study has received approval from the Ethics Committee of the Medical University of Warsaw. Results will be submitted to a peer-reviewed journal and presented at national and international scientific meetings.</p> Trial registration <p>NCT06448767. Registered on 3 June 2024</p> <p>Protocol version</p> <p>Version 2, 27 of October 2025</p>