In this study, the levels of concentration of carboxylic acids (benzoic acid, p-hydroxybenzoic acid, p-hydroxyphenylacetic acid, and hippuric acid) in the urine of autistic children were investigated and compared. The increased excretion of carboxylic acids is related to excessive bacterial activity in the gut, called bacterial overgrowth, which has been related to autism spectrum disorder (ASD) as an impairment in the gut-brain axis. The investigation was based on the analysis of urine samples obtained from 120 ASD children. To identify and quantify urinary carboxylic acids (UCAs), we applied gas chromatography coupled with mass spectrometry (GC-MS). Additionally, we checked the influence of probiotic supplementation, gender, body mass index (BMI) value and age of children on the level of different selected compounds. Most of the obtained results were found within reference ranges. In some cases, the levels of benzoic acid, p-hydroxybenzoic acid, and p-hydroxyphenylacetic acid were particularly elevated. Statistically significant differences were observed in supplementation with probiotics and the level of p-hydroxyphenylacetic acid (p=0.036). The obtained results may indicate disturbances in the intestinal flora in some autistic patients and suggest that supplements may have an influence on the levels of carboxylic acids in urine. Due to the small population of children taking the supplement, further study are needed.