Background/Objectives: The study of microbiome composition shows positive indications for application in the diagnosis and treatment of many conditions and diseases. One such condition is autism spectrum disorder (ASD). We aimed to analyze gut microbiome samples from children in Bosnia and Herzegovina to identify microbial differences between neurotypical children and those with ASD. Additionally, we developed machine learning classifiers to differentiate between the two groups using microbial abundance and predicted functional pathways. Methods: A total of 60 gut microbiome samples (16S rRNA sequences) were analyzed, with 44 from children with ASD and 16 from neurotypical children. Four machine learning algorithms (Random Forest, Support Vector Classification, Gradient Boosting, and Extremely Randomized Tree Classifier) were applied to create eight classification models based on bacterial abundance at the genus level and KEGG pathways. Model accuracy was evaluated, and an external dataset was introduced to test model generalizability. Results: The highest classification accuracy (80%) was achieved with Random Forest and Extremely Randomized Tree Classifier using genus-level taxa. The Random Forest model also performed well (78%) with KEGG pathways. When tested on an independent dataset, the model maintained high accuracy (79%), confirming its generalizability. Conclusions: This study identified significant microbial differences between neurotypical children and children with ASD. Machine learning classifiers, particularly Random Forest and Extremely Randomized Tree Classifier, achieved strong accuracy. Validation with external data demonstrated that the models could generalize across different datasets, highlighting their potential use.