Enterobacteriaceae are the leading cause of urinary tract infections, and include pathogens such asE. coli, K. pneumoniae and P. mirabilis. Due to their similarity, the correct identification of these pathogensis difficult and time-consuming. Raman spectroscopy has been demonstrated extensively as a tool forrapid microbiological differentiation. However, for pathogenic Enterobacteriaceae the application ofRaman spectroscopy has been particularly challenging. In this study, two promising methods for Ramanbasedmicrobiological diagnostics were compared for differentiating Enterobacteriaceae. Spectra werecollected from single-cells with Raman microspectroscopy and from colonies on agar with an NIR Ramanfiber-probe. A comprehensive dataset of spectra from 8 different, clinically relevant, genera was collected.Visually, the spectra obtained from both methods presented little difference between the genera. Forclassification, single cell analysis yielded limited results, while the fiber-probe spectra enabled perfectclassification of all 16 isolates. Moreover, the model was validated on new replicates and 15/16 strainswere correctly identified (94% overall accuracy). This is the first study to focus on the closely relatedEnterobacteriaceae, who have previously been avoided or differentiated poorly. It shows how, with thecorrect spectroscopic setup, even challenging questions in clinical microbiology can be resolved withRaman spectroscopy, highlighting the method’s potential for improving patient care.