Research involving bacterial pathogens often requires enumeration of bacteria colonies. Here, we present a low-cost, high-throughput colony counting system consisting of colony counting software and a consumer-grade digital camera or document scanner. We demonstrate that this software, called "NICE" (NIST's Integrated Colony Enumerator), can count bacterial colonies as part of a high-throughput multiplexed opsonophagocytic killing assay used to characterize pneumococcal vaccine efficacy. The results obtained with NICE correlate well with the results obtained from manual counting, with a mean difference of less than 3%. NICE is also rapid; it can count colonies from multiple reaction wells within minutes and export the results to a spreadsheet for data processing. As this program is freely available from NIST, NICE should be helpful in bacteria colony enumeration required in many microbiological studies, and in standardizing colony counting methods. © 2010 International Society for Advancement of Cytometry.