Chromoblastomycosis is a chronic subcutaneous disease caused by human contact with melanized fungioccurring mainly in tropical and subtropical zones worldwide. This study assessed 12 patients with chro-moblastomycosis from Rondˆonia, Brazil, Amazon region. In sum, 83.3% were men, 41.6% were from MonteNegro city, median age was 52.9 years, and median time to disease progression was 12.2 years. Lesions werelocated on the lower limbs (75%), and verruciform was prevalent form (66.6%). After 3 years of treatmentwith itraconazole, two patients were considered cured. The etiological agents were identified by the molec-ular sequence of the ribosomal internal transcribed spacer ITS1, 5.8S, and ITS2 region andβ-tubulin genes.Eight strains were identified asFonsecaea pedrosoi, two wereF. nubica,and two wereRhinocladiella similis.The antifungal activity of five drugs was evaluated, and the most active drug was terbinafine (range minimalinhibitory concentration [MIC] 0.015–0.12μg/ml), itraconazole (range MIC 0.03–0.5μg/ml) and voriconazole(range MIC 0.06–0.5μg/ml). The highest MIC was 5-fluorocytosine (range MIC 2–32μg/ml), and ampho-tericin B (range MIC 0.25–2μg/ml). In conclusion, the present study expanded the epidemiological diseasedatabase and described for the first timeF. nubicaandR. similisas chromoblastomycosis agents in theBrazilian Amazon region. Our results confirmed the importance of using molecular methods to identify themelanized fungi and stimulate the recognition of the disease in other places where no cases have beenreported.