Background: The hypothesis that early stimulation of the gut microbiota contributes to immune system balance has encouraged the use of probiotics to treat atopic dermatitis (AD), an immunological disorder characterized by chronic and relapsing skin inflammation, in several clinical studies. This study aimed to evaluate the clinical efficacy of a mixture of probiotics (Lactobacillus and Bifidobacterium) in children and adolescents with AD and the effects of probiotics on sensitization, inflammation, and immunological tolerance. Methods: In this double-blind, randomized, placebo-controlled clinical trial, children and adolescents (aged 2 to 19 years) received one gram (sachet) per day of probiotics or placebo for six months. SCOring of AD (SCORAD) index, serum IgE levels, skin prick test, tolerogenic and inflammatory cytokines were evaluated. Results: Forty patients were evaluated and clinical response was significantly better in the probiotic group as compared to the placebo group after treatment for six months; SCORAD decreased (95% CI, 2.44–52.94) even after adjustment for co-variables (95% CI, 5.52–59.13). Three months after the treatment was discontinued, improvement persisted in the probiotic group even after adjustment for co-variables (95% CI, 0.78– 27.70). IgE levels, skin prick test and cytokines did not differ between groups. Conclusions: AD children and adolescents treated with a mixture of probiotics (Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus paracasei, and Bifidobacterium lactis) for six months presented a significate SCORAD reduction as compared to placebo group. This probiotics mixture did not affect SPT and IgE levels, as well as inflammatory or tolerogenic cytokines. #NCT02519556.