We recently suggested that oral malodor production involves two steps: (i) deglycosylation of glycoproteins and (ii) proteolysis and amino acid utilization of the protein core to yield volatiles such as volatile sulfide compounds (VSCs). Our aim was to test the hypothesis that β-galactosidase activity and VSC production occur in distinct areas of the biofilm by two different bacterial populations. Biofilms were grown anaerobically for seven days with or without antibiotics (i.e. vancomycin and metronidazole). Biofilms were stained for β-galactosidase activity and VSC production and studied using confocal laser scanning microscopy. Results showed that β-galactosidase activity occurs in the outer layers and disappears following vancomycin addition, whereas VSC production occurs deeper within the biofilm and disappears following metronidazole application. These findings suggest that β-galactosidase activity is produced mainly by Gram-positive oral bacteria at the outer portion of the biofilm, and VSC production occurs in the deeper layers by Gram-negative oral bacteria.