Bovine brucellosis is a disease affecting cattle worldwide and is mainly caused by Brucella abortus. Diagnosis of brucellosis is an important part of controlling bovine brucellosis in naturally infected cattle in South Africa. Serology remains the most practical method available to screen herds and confirm diagnosis. This study investigated the performance of Rose Bengal test (RBT), complement fixation test (CFT), serum agglutination test (SAT), competitive enzyme-linked immunoabsorbent assays (cELISA), indirect ELISA (iELISA) and B. abortus species specific (BaSS) PCR in the diagnosis of brucellosis in naturally infected cattle in Kwazulu-Natal Province of South Africa. Natural brucellosis infection status of animals was determined by culturing from abomasal fluid, milk, hygroma fluid, lymph nodes or uterine discharges samples. Some of the samples were used in the BaSS PCR assay whereas sera obtained from the same animals were tested using RBT, CFT, SAT, cELISA and iELISA. Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were determined for each test to assess each test performance to determine bovine brucellosis in naturally infected animals. Positive cultures were identified as B. abortus biovar 1. The iELISA and RBT had the highest DSe of 91.7% (95% confidence interval (CI): 80.0-97.7%) followed by cELISA with a DSe of 91.5% (95% CI: 79.6-97.6%) and the CFT and SAT with DSe values of 91.4% (95% CI: 79.6-97.6%) and 87.2% (95% CI: 74.3-95.2%) respectively. The BaSS PCR had the lowest DSe of 72.7% (95% CI: 39.0-94.0%), but highest DSp of 92.0% (95% CI: 79.1%-98.4%) followed by the RBT and iELISA DSp value of 86.7% (95% CI: 55.5%-98.3%). CFT and cELISA had a DSp of 81.3% (95% CI: 68%-89%) whilst the SAT had the lowest DSp of 68.8% (41.3%-89.0%). Although there was no statistically significant difference between the six tests in their ability to diagnose brucellosis, the RBT and indirect ELISA had the highest diagnostic sensitivity and specificity. The BaSS PCR diagnosed B. abortus at species level and could be used to confirm culture results.