We used droplet digital PCR (ddPCR) assays to detect/quantify DNA from Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus spp. in blood samples. Bacterial DNA from clinical strains (4 < n < 12) was extracted, quantified and diluted (10-0.0001 ng/µL) and ddPCR assays were performed in triplicate. These ddPCR assays showed low replication variability, low detection limit (1-0.1 pg/µL), and genus/species specificity. ddPCR assays were also used to quantify bacterial DNA obtained from spiked blood (1 × 10 -1 CFU/mL) of each bacterial genus/species. Comparison between ddPCR assays and bacterial culture was performed by Pearson correlation. There was an almost perfect correlation (r ≥ 0.997, P ≤ 0.001) between the number of CFU/mL from bacterial culture and the number of gene copies/mL detected by ddPCR. The time from sample preparation to results was determined to be 3.5 to 4 hours. The results demonstrated the quantification capacity and specificity of the ddPCR assays to detect/quantify 4 of the most important bloodstream infection (BSI) bacterial pathogens directly from blood. SIGNIFICANCE AND IMPACT: This pilot study results support the potential of ddPCR for the diagnosis and/or severity stratification of BSI. Applied to patients' blood samples it can improve diagnosis and diminish sample-to-results time, improving patient care.