Deregulation of micro-RNAs (miRNAs) may contribute to mechanisms of injury in the bicuspid aortic valve (BAV). Our objective was to investigate the expression of miRNAs in aortic tissue from patients who underwent aortic valve replacement for aortic stenosis and its relationship with aortic dilatation. The study included 78 patients, 40 with bicuspid aortic valve (BAV) and 38 with tricuspid aortic valve (TAV). The expression of miRNA-17-5p, hsa-let-7e, and miRNA-196a-5p in human aortic tissue was evaluated by a reverse transcriptase polymerase chain reaction (RT-qPCR). Comparative analysis between patients with BAV and controls with TAV explored the association between the miRNAs and aortic dilatation (AD), calcification, valve dysfunction, and stenosis. The results showed that the expression levels of miRNA-Let-7e-5p and miRNA-196-5p were mostly increased in patients with BAV and aortic dilatation ( = 0.01 and = 0.01), respectively. In contrast, the levels of miRNA-17a-5p ( < 0.20) were lower but without a statistically significant difference. The downregulation of miRNA-17a-5p and the upregulation of miR-Let-7e-5p and miR-196-5p were related to an increased risk of AD risk. Subjects with BAVs with or without double aortic lesions had higher expression levels of Let-7e-5p and miRNA-17a-5p vs. TAV. In all patients, we found an inverse correlation of MiRNA-196-5p with High-Density Lipoprotein-Cholesterol (HDL-C) and indexed valvular area. In subjects with a higher expression of miRNA196, lower levels of HDL-C correlation (r) [r 0.27 ( = 0.02)] and a lower indexed valvular area [r 0.28 ( = 0.05)] were observed. In the specific analysis for each patient group, it was found that in control subjects with tricuspid aortic valve (TAV), miRNA-196-5p had a positive correlation with valvular calcification (r = 0.60, = 0.02). Deregulation of miRNAs in the aortic tissue of a BAV may influence valvular stenosis, dysfunction, and concomitant aortic dilation. This information could help to define potential therapeutic target strategies to improve the prognosis and treatment of BAV.