We report a novel workflow for the isolation of bacteria from whole blood samples. We detected E. coli from 50 μL of whole blood in concentrations as low as 400 CFU/mL in less than 1 h using a microfluidic device. The method presented consists of reducing the number of blood cells found in whole blood by selectively lysing the blood cells with a solution of saponin and sodium cholate. We demonstrate that the bacteria grow during the time of loading in the microchip and can continue to be grown afterwards on the chip. Additionally, we show our approach is compatible with some of the most common sepsis-causing bacteria. The method can half the bacterial culture time needed for antibiotic susceptibility testing (AST) of bloodstream infections.