Rapid diagnostic tests that differentiate between Gram positive, Gram negative and the absence of aerobic bacteria in milk samples from dairy cows with clinical mastitis can support antimicrobial treatment decisions and contribute to a more prudent use of antimicrobials in the dairy industry. The objective of this study was to evaluate the test characteristics of the novel rapid BACT mastitis test in discriminating causes of clinical mastitis under laboratory conditions. Test outcomes of 155 milk samples from clinical mastitis cases were incubated for 14–16 h in the BACT test and compared to results of bacteriological culture. The accuracy for detection of bacterial growth and Gram positive growth was 91 and 89%, respectively. The BACT test could provide an accurate and relatively fast decision tool for farmers to aid in antimicrobial treatment decisions in cases of clinical mastitis.