Milk quality in bulk tank milk (BTM) is measured by flow cytometry technology as total bacterial count (TBC) and somatic cell count (SCC). To investigate SCC problems, culture or PCR can be used to identify mastitis causing bacteria, e.g., Mastit 4, a commercially available qPCR test. TBC in BTM can be investigated further using culture-based methods such as standard plate count, laboratory pasteurization count, coliform count, and spore counts. To our knowledge, no qPCR addressing the bacteria involved in TBC has been commercially introduced. The aim of this study is to evaluate a recently introduced 3-h qPCR test, TBC 4. The TBC 4 qPCR detects four target groups, Pseudomonas, Streptococci, Enterobacteriacea/Enterococcus, and Bacillus/Clostridia. These target groups relate to problems on the farm such as cooling, mastitis, environment, and silage. We will continue with new research to compare the TBC 4 qPCR test with traditional culture. For this study, BTM samples from different TBC intervals were selected based on BactoCount results found at routine payment investigation at Eurofins laboratory (Vejen, Denmark). These samples were analyzed using TBC 4 qPCR assay within 24 h. In total, 346 BTM samples were divided into six different intervals of colony forming units (CFU). For all four targets in each of the different intervals of CFU, the percent of positive samples, the average Ct-value, the percent of positive samples with Ct<30 and Ct<25 were calculated. For Pseudomonas, Streptococci, and Enterobacteriacea/Enterococcus, the number of positive samples with lower Ct-values (high bacteria content) correlated with the CFU mL–1. We found Enterobacteriacea/Enterococcus, Pseudomonas, and Streptococci in high number of bacteria (Ct<25) in 25, 19 and 56% of samples with CFU mL–1 between 50 001–100 000 and 53, 44, and 39% in samples with CFU mL–1>100 000. The TBC 4 qPCR test showed to be a strong and fast tool for farmers, advisors and service technicians to address problems with high TBC and ensuring the delivery of good quality milk to the dairy.