This study aimed to investigate the prevalence of human brucellosis in Najran, southwestern Saudi Arabia, and to assess the performances of ELISA and PCR as diagnostic tools for brucellosis with respect to conventional methods. The study included 340 patients with clinical characteristics of brucellosis. Blood samples from cases and controls were subjected to culture, standard tube agglutination test (SAT), ELISA for IgM and IgG, and brucella PCR. The diagnosis of brucellosis was confirmed in 54 (15.9%) of the 340 provisionally diagnosed brucellosis patients. Blood culture identified only 14 (25.9%) cases. The SAT was positive for 50 (92.6%) cases, whereas ELISA IgM, IgG and PCR were found positive in 46, 52 and 38 cases respectively. The sensitivities of ELISA IgM and IgG were 85.2% and 96.3% respectively and the specificity was 100% for each. For PCR, the sensitivity and specificity were 70.4% and 100% respectively. In conclusion, ELISA offers a significant advantage over conventional serological methods in the diagnosis of brucellosis in endemic areas. The PCR test results can be particularly important in patients with clinical signs and symptoms, and negative serological results, allowing the early and rapid confirmation of the brucellosis.