Currently available bacterial source-tracking tools are often technically demanding, time consuming, and have limited accuracy in grouping isolates according to their respective sources. There is a need for the development of bacterial source-tracking tools that would allow for more rapid and accurate grouping of isolates by source. We examined the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the characterization of environmental isolates of Enterococcus. Our main objectives were to develop sample preparation protocols for obtaining reproducible MALDI-TOF mass spectra from Enterococcus isolates and to evaluate methods of data analysis to maximize repeatability of the method and its ability to group isolates according to their respective sources. Our data showed that treatment of 21 Enterococcus isolates from seven unique sources with lysozyme for 20 h, followed by calculation of similarity coefficients using the Pearson product-moment correlation coefficient, facilitated a repeatability level of 91% as well as grouping by source for isolates obtained from several sources including human waste. Our data suggest that MALDI-TOF-MS-based fingerprinting of environmental isolates of Enterococcus has potential as a rapid and accurate bacterial source tracking (BST) tool, but requires further development, specifically regarding the time requirements needed for pre-treatment of isolates with lysozyme.