This study aimed to investigate the gut microbiota composition in children with autism spectrum disorder (ASD) compared to neurotypical (NT) children, with a focus on identifying potential differences in gut bacteria between these groups. The microbiota was analyzed through the massive sequencing of region V3–V4 of the 16S RNA gene, utilizing DNA extracted from stool samples of participants. Our findings revealed no significant differences in the dominant bacterial phyla (Firmicutes, Bacteroidota, Actinobacteria, Proteobacteria, Verrucomicrobiota) between the ASD and NT groups. However, at the genus level, notable disparities were observed in the abundance of Blautia, Prevotella, Clostridium XI, and Clostridium XVIII, all of which have been previously associated with ASD. Furthermore, a sex-based analysis unveiled additional discrepancies in gut microbiota composition. Specifically, three genera (Megamonas, Oscilibacter, Acidaminococcus) exhibited variations between male and female groups in both ASD and NT cohorts. Particularly noteworthy was the exclusive presence of Megamonas in females with ASD. Analysis of predicted metabolic pathways suggested an enrichment of pathways related to amine and polyamine degradation, as well as amino acid degradation in the ASD group. Conversely, pathways implicated in carbohydrate biosynthesis, degradation, and fermentation were found to be underrepresented. Despite the limitations of our study, including a relatively small sample size (30 ASD and 31 NT children) and the utilization of predicted metabolic pathways derived from 16S RNA gene analysis rather than metagenome sequencing, our findings contribute to the growing body of evidence suggesting a potential association between gut microbiota composition and ASD. Future research endeavors should focus on validating these findings with larger sample sizes and exploring the functional significance of these microbial differences in ASD. Additionally, there is a critical need for further investigations to elucidate sex differences in gut microbiota composition and their potential implications for ASD pathology and treatment.