Brucellosis is a zoonotic disease in humans with its public health importance. Laboratory diagnostic methods targeting brucellosis are not performed in hospital settings across Ghana. Very little is known about the comparative diagnostic abilities of the various tests available presently. The aim of this study therefore was to evaluate and compare diagnostic performances of Rose Bengal Plate Test (RBPT), Enzyme Linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR) employed in diagnosing Brucella infection. Two hundred and twenty Abattoir workers were randomly selected in Kumasi, Ghana. Blood samples were collected, serum extracted and tested for the presence of anti-Brucella antibodies and compared among three different techniques, using ELISA, RBPT and PCR. From the 220 participants tested for antibodies against Brucella spp., 3 (1.4%), 4 (1.8%) and 21 (9.6%) were positive for Rose Bengal Plate test, anti-Brucella ELISA IgM and anti-Brucella ELISA IgG respectively. A total of 98 (44.5%) participants tested positive by PCR. The sensitivity, specificity, positive predictive value, negative predictive values and Kappa value for Rose Bengal in comparison with PCR were 66.7%, 55.8%, 2.0%, 100% and 0.013 respectively while that for ELISA IgG in comparison with PCR were 85.7%, 71.3%, 18.4%, 98.5% and 0.212 respectively. PCR yielded the highest sensitivity and specificity among the three diagnostic methods in this study and should be considered for use at strategic reference laboratories to augment existing routine serological tests for brucella performed in laboratories in Ghana.