Bladder cancer is the second most common cancer in the genitourinary tract, showing often recurrence and progress into invasive states. Epigenetic changes, such as microRNA alteration are involved in bladder cancer tumorigenesis through a variety of signaling pathways. The epigenetic state depends on geographic and lifestyle conditions. The aim of this study was to investigate the expression level of microRNA-99a and microRNA-205 in bladder cancer in Iranian populations and to determine the relationship between their expressions with clinicophatological features.36 patients with bladder cancer were included in the study. The control group was the healthy adjacent tissue of the same patients. Total RNA was extracted from approximately 50 mg tissue using TRIzol reagent. cDNA was synthesized and Real-Time PCR was carried out using specific primers. The Unisp6 rRNA was used as a reference gene. A significant decrease was found in the expression level of miR-99a in tumor samples, compared to healthy adjacent tissues (P<0.001). The increased expression level of miR-99a was significantly associated with muscle invasion (P=0.02). The receiver operating characteristic (ROC) analysis for miR-99a showed AUC value equal to 0.944, with specificity of 97%, sensitivity of 91%, and cut off value of 8.31 (P<0.001). A significant association was found between smoking and miR-99a (P=0.04) and miR-205 (P= 0.01) expression levels. Dramatic down-regulation of miR-99a in bladder cancer tissues confirmed the tumor suppressor role of miR-99a in bladder cancer. A higher amount of miR-99a expression was associated with invasive bladder cancer. According to ROC analysis, miR-99a could be considered as a valuable diagnostic biomarker.