Recurrent aphthous stomatitis (RAS) is a common inflammatory disorder of the oral mucosa characterized by recurrent, painful ulcerations with multifactorial etiology. Emerging evidence suggests that oral microbial dysbiosis and antimicrobial resistance may contribute to lesion persistence and recurrence. The present study aimed to investigate the microbiological and molecular characteristics of oral microflora associated with RAS. Oral swab samples (n = 13) were collected from patients with active aphthous ulcers and subjected to culture-based identification, Gram staining, and biochemical characterization. Antimicrobial susceptibility testing was performed using the Kirby–Bauer disc diffusion method. Phenotypic detection of extended-spectrum β-lactamase (ESBL) production was carried out using the combination disc method, followed by PCR-based detection of resistance genes. The results demonstrated a predominance of Staphylococcus aureus and Pseudomonas aeruginosa in ulcerative lesions. Gram-positive isolates exhibited resistance to oxacillin and ciprofloxacin, while Gram-negative isolates showed multidrug resistance patterns. Notably, 50% of P. aeruginosa isolates were confirmed as ESBL producers. Molecular analysis identified the presence of blaTEM and blaCTX-M genes among phenotypically positive isolates. These findings highlight a significant association between oral microbial dysbiosis, antimicrobial resistance, and RAS, underscoring the importance of microbiological profiling in understanding disease pathogenesis and guiding targeted therapeutic strategies