Cholesteatoma, a destructive middle ear condition, poses challenges due to its variable clinical presentation and propensity for recurrence. Understanding its molecular underpinnings could enhance prognostication and guide therapeutic interventions. This study investigates the association between cholesteatoma aggressiveness, as assessed by the Middle Ear Risk Index (MERI), and the expression of miRNA-21 and IL-6 genes. A cross-sectional observational study involving 30 patients with cholesteatoma undergoing tympanomastoid exploration was conducted. MERI scores were calculated preoperatively, and cholesteatoma tissue was analyzed for miRNA-21 and IL-6 gene expression using RT-PCR. Statistical analysis was performed to correlate MERI scores with gene expression levels. The majority (80%) of patients exhibited severe MERI scores, correlating with extensive middle ear pathology and necessitating canal wall-down (CWD) mastoidectomy. Higher miRNA-21 and IL-6 gene expression levels were observed in cholesteatoma tissues, indicating local aggressiveness and inflammatory activity. Significant moderate correlations were found between MERI scores and miRNA-21 (Pearson correlation = 0.579, p = 0.001) and IL-6 gene expression (Pearson correlation = 0.388, p = 0.034). Patients with severe MERI scores had elevated miRNA-21 and IL-6 levels, suggesting a more aggressive disease phenotype. MERI scores demonstrated utility in predicting cholesteatoma aggressiveness, with higher scores correlating with elevated miRNA-21 and IL-6 expression. These findings suggest a potential role for MERI in guiding surgical decision-making and prognostication. Future research on targeted therapies based on molecular mechanisms holds promise for improving cholesteatoma management. 3 Laryngoscope, 2024.