Purpose: Clinical studies with dentifrices formulated with antimicrobial ingredients typically examine the effects of their use on clinical parameters of dental plaque and gingivitis. The effects of routine use of these formulations on the microorganisms found in the distinct regions of the human mouth remain unexplored. The present cross-over design clinical study compared the effects of brushing with a recently formulated dentifrice with 1% zinc citrate to a control dentifrice on oral bacteria found in oral niches. Materials and Methods: A total of 35 adults completed a 1-week wash-out phase prior to providing baseline samples of dental plaque on the teeth, gums, saliva and scrapings from cheeks and tongue to quantify anaerobic bacteria. Subjects brushed twice daily with a randomly assigned test dentifrice for the next 13 days. On the 14th day, they arrived at the dental clinic prior to oral hygiene and provided samples similar to baseline samples for microbiological examinations. Subjects brushed with the test formulation they used in the previous 13 days, prior to providing additional samples at 5-h post-brushing for microbial assessments. An additional wash-out phase was assigned to subjects prior to completing the study with the alternate test dentifrice. Results: Analyses of log10-transformed microbial counts indicated no statistical differences in the baseline samples from each oral site (P > 0.05), while brushing with the zinc citrate dentifrice resulted in a significant reduction in bacteria in each post-brushing oral site versus the control paste (P 0.05). Brushing with the zinc citrate dentifrice resulted in 38.56%, 23.57%, 26.24% and 29.1% decreases in anaerobic bacteria from dental plaque, tongue, cheek and saliva, respectively, in the overnight samples collected on the 14th day versus the control. Similarly, the 5-h post-brushing samples indicated 24.8%, 34.25%, 37.98% and 38.31% reductions in anaerobic organisms when compared with control. Conclusions: Toothbrushing with the zinc citrate formulation resulted in significant reductions in oral bacteria from all of the intraoral locations. Keywords: adults, anaerobic, bacteria, cheek, clinical study, dental plaque, fluoride, tongue, zinc citrate