The aim of this study was to evaluate the whole-blood levels of miR-155, miR-223, miR-181a, miR-146a, and miR-let-7e in patients with bilateral knee osteoarthritis (OA) after a cycle of mud-bath therapy (MBT). Thirty-two patients with knee OA defined by the ACR criteria were included. Twenty-one patients (MBT group) were daily treated with a combination of local mud-packs at 42 °C and baths in mineral water, at 37 °C for 15 min, for 12 applications over a period of 2 weeks, in addition to standard therapy; 11 patients (control group) continued their conventional treatment alone. Global pain score evaluated by visual analog scale (VAS), WOMAC subscores, and microRNA expression were evaluated at baseline and after 2 weeks. Peripheral whole blood was collected into PAXgene™ Blood RNA tubes, stored at - 80 °C, and total RNA was extracted. The expression of miR-155, miR-223, miR-181a, miR-146a, and miR-let-7e was determined by qRT-PCR. After MBT, we observed a statistically significant improvement of clinical parameters and a significant decrease of miR-155, miR-181a, miR-146a (p < 0.001), and miR-223 (p < 0.01) expression levels. No clinical and biochemical modifications were detected in the control group. No significant variations of miR-let-7e were shown in both groups after 2 weeks. In conclusion, MBT can modify the expression of miR-155, miR-181a, miR-146a, and miR-223, which are upregulated in OA. It could be due to the heat stress and the hydrostatic pressure, since some miRNAs were found to be temperature- and mechano-responsive. Further studies are needed to better explain the mechanism of action of MBT and the role of miRNAs in OA.