The identification of pathogens in ascitic fluid is standardly performed by ascitic fluid culture, but this standard procedure often needs several days. Additionally, more than half of the ascitic fluid cultures are negative in case of suspected spontaneous bacterial peritonitis (SBP). It is therefore important to identify and characterize the causing pathogens since not all of them are covered by the empirical antimicrobial therapy. The aim of this study is to show that pathogen identification in ascitic fluid is possible by means of Raman microspectroscopy and chemometrical evaluation with the advantage of strongly increased speed. Therefore, a Raman database containing more than 10000 single-cell Raman spectra of 34 bacterial strains out of 13 different species was built up. The performance of the used statistical model was validated with independent bacterial strains, which were grown in ascitic fluid.