Background: Human brucellosis, also known as Malta fever, is an acute systemic zoonotic disease in several parts of the world. The most pathogenic Brucella specie is Brucella melitensis that occurs in the human population of all age groups and of both sexes. Objectives: The present study aimed to investigate human Brucella infection in Afghanistan. Methods: The participants in this cross-sectional study were 44 patients diagnosed with Brucella infection during eight months and confirmed using Wright test by physicians of Iran Clinic Hospital. For data analysis, a statistical model was used through SPSS. Results: The most affected patients were female housewives (40.9%) and students (18.18%). The most frequent clinical manifestations were recurrent attacks of fever (95.34%), weight loss (81.39%), loss of appetite (79.06%), musculoskeletal pain (69.76%), boredom (67.44%), and lethargy (60.46%). A total of 41 (93.18%) patients mentioned the consumption of unpasteurized milk as the source of infection. Conclusions: The study results revealed that the main route of Brucella transmission in Afghanistan is the consumption of contaminated dairy products. The highest prevalence of brucellosis was observed among the young and middle-aged populations and housewives.