Early identification of the causative pathogens in physiological specimens that require no cultivation is essential for directing an evidence-based antimicrobial therapy in resource limited settings. Here, we describe an integrated microfluidic device for rapid identification of pathogens in complex physiological matrices such as blood. The device was designed and fabricated using SlipChip technologies, which integrated four channels processing independent samples and identifying up to twenty different pathogens. Briefly diluted whole human blood samples were directly loaded into the device for analysis. Pathogens were derived from blood by dielectrophoresis, retained in the array of grooves, and identified by multiplex array PCR in nanoliter volumes with end-point fluorescence detection. The universality of dielectrophoretic separation of pathogens from physiological fluids was evaluated with a panel of clinical isolates covering predominant bacterial and fungal species. Using this system, we simultaneously identified Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli O157:H7 within 3 hours. In addition to the prompt diagnosis of bloodstream infections, this method may be also utilized for differentiating microorganisms in other physiological and environmental samples.