Background:Sepsis is a common serious condition worldwide and diagnostics are still slow. Nanopore sequencing is a promising tool in point of care medicine. However, high ratio of host to bacterial DNA decreases utility of nanopore. Fast, cheap, and easy techniques to separate host from bacterial DNA are required. Aim:The aim was to compare three different separation methods of blood for bacterial DNA enrichment and isolation with subsequent products readily suitable for nanopore sequencing. Methods: Blood was collected from healthy subjects and spiked with either gram-negative or gram-positive bacteria. Three different separation methods alongside negative control were performed. The performance of each method was evaluated by RT-qPCR of 16S gene and B2 microglobulin. Results: 16s RT-qPCR showed similar Ct value for tested methods in comparison to Standard method.However, the host DNA depletion was significantly higher for the centrifugal method compared to others. Reproducibility, based on variability measured by the standard deviation and RT-qPCR results, was highest using the centrifugation method compared to the other three methods. Conclusion: From all tested methods, centrifugation outperformed other basic isolation techniques considering turnaround time, reliability, and effectiveness. Further investigation is needed to confirm our promising results in a real-life scenario.