Objective: This study aimed to evaluate the impact of orthodontic treatment on the oral microbiota and oral health outcomes in pediatric patients, focusing on changes in microbial diversity, salivary pH, and clinical parameters such as gingival and plaque indices. Methods: A longitudinal observational study was conducted at Bacha Khan College of Dentistry from January 2023 to January 2024, involving 78 pediatric patients aged 6–16 years. Participants underwent orthodontic treatment with fixed braces, aligners, or functional appliances. Saliva and plaque samples were collected at baseline (pre-treatment) and six months into treatment. Microbial diversity was assessed using 16S rRNA sequencing, while Streptococcus mutans and Lactobacillus colony-forming units (CFUs) were quantified through microbial culturing. Salivary pH and clinical indices, including gingival index (GI), plaque index (PI), and bleeding on probing (BOP), were recorded and analyzed. Results: The Shannon Diversity Index decreased significantly during treatment (p = 0.002), indicating reduced microbial diversity. Levels of S. mutans and Lactobacillus increased significantly (p < 0.05), while salivary pH decreased (p = 0.012), reflecting an acidogenic shift. Clinical indices showed a significant rise in plaque accumulation and gingival inflammation, with GI, PI, and BOP all worsening during treatment (p < 0.05). Caries incidence increased from 8% to 15% over six months. Conclusion: Orthodontic treatment significantly impacts oral microbiota and oral health in pediatric patients, reducing microbial diversity, increasing cariogenic bacteria, and heightening the risk of gingival inflammation and caries. These findings underscore the importance of preventive strategies, including enhanced oral hygiene education, regular professional cleanings, and fluoride and antimicrobial products, to mitigate treatment-related complications.