Previous research has shown that the production of volatile sulfide compounds (VSC) by oral bacteria is associated with oral malodor. In the present study, we report a novel technique (microscopic sulfide assay (MSA)) for the quantification of VSC-producing oral microorganisms. The MSA was performed by overnight incubation of saliva samples in the presence of ferrous sulfate and sodium thiosulfate, followed by digital analysis of cells stained black due to cell-associated precipitation of ferric sulfide. This method was found to correlate significantly with oral malodor parameters, including mean odor judge scores (two judges, r = 0.48 and p = 0.001) and Halimeter® readings (r = 0.53 and p < 0.001), in a group of 42 subjects. As compared with odor judge scores as the gold standard, the new MSA technique yielded a diagnostic accuracy of 0.7 (ROC, p = 0.023). Results indicate that the MSA may serve as a diagnostic technique for assessing oral malodor levels and aid in identifying the particular bacteria involved in this condition.