Objective: This study aimed to evaluate the impact of orthodontic appliances on the oral microbiota of pediatric patients with pre-existing pathological conditions, focusing on microbial diversity, biofilm formation, and oral health outcomes. Methods: A total of 110 pediatric patients aged 6–12 years were enrolled in this prospective observational study conducted at Khyber Medical University from January 2023 to January 2024. Participants were categorized based on the type of orthodontic appliance used (fixed or removable). Plaque and saliva samples were collected at baseline and follow-up visits and analyzed for microbial composition, biofilm density, salivary pH, and periodontal indices. Data were statistically analyzed, with p-values <0.05 considered significant. Results: Fixed appliances led to a significant increase in pathogenic bacterial species, including Streptococcus mutans and Lactobacillus, compared to removable appliances (p < 0.05). Biofilm thickness nearly doubled, and salivary pH decreased significantly from baseline (7.1 ± 0.2) to follow-up (6.8 ± 0.3). Children with pre-existing conditions, such as asthma and diabetes, exhibited heightened gingival inflammation and caries risk. Gingivitis was the most prevalent complication (54.5%), followed by caries (31.8%) and enamel decalcification (22.7%). Conclusion: Orthodontic appliances, particularly fixed types, significantly alter the oral microbiota and increase the risk of oral health complications in pediatric patients with systemic health conditions. Preventive strategies, including enhanced oral hygiene practices and regular professional care, are essential to mitigate these risks.