Objective: To determine epidemiological, molecular characterization, and potential risk factors of human brucellosis. Methods: This descriptive study was carried out in the clinical setting in Iran between 2017 and 2018. A total of 297 participants enrolled in the study. The sample size was calculated based on the occurrence rate of brucellosis in different areas. Patients were assessed using serological tests and conventional culture methods. Phage and multiplex PCR methods typed all of Brucella isolates. Potential risk factors of disease were determined. Results: A total of 141 of 297 (47.5%) Brucella strains were isolated and all of them were detected as Brucella melitensis biovar 1. Based on serologic titers, high culture positivity was recorded at 1/640 titer (P< 0.006). The risk factors for brucellosis were patients older than 40 years (OR=2.23, 95%CI: 1.4-3.55, P=0.001), animal keeper (OR=7, 95%CI: 1.51-32.41, P=0.005), housewife (OR=8.76, 95%CI: 1.85-41.37, P=0.002), farmer (OR=6.42, 95%CI: 1.21-33.97, P=0.019), and contact with animal (OR=1.31, 95%CI: 0.60-2.85, P=0.005). Conclusions: To the best of our knowledge, this is the first comprehensive report from Iran presenting the detection of Brucella species by the multiplex PCR. Brucella melitensis biovar 1 is still the dominant causative agent in Iran. The consumption of unpasteurized dairy products, living in rural areas, and animal contact were risk factors of brucellosis.