It is generally recommended that toothbrushes should be replaced after three-months' use in order to maintain efficacy. This clinical investigation employed a single-use, cross-over study and a three-month parallel-group study to investigate the effect of toothbrush wear on plaque and gingival health. Toothbrushes were artificially worn using a laboratory wear machine to simulate three months of clinical toothbrush use. Results from the single-use study showed that both the new and the worn toothbrushes significantly reduced whole mouth, marginal and approximal plaque scores from pre- to post-brushing (p < 0.0001). The new brush was, however, significantly more effective than the worn brush, demonstrating 13.4%, 11.0%, and 17.0% greater plaque reduction for whole mouth, marginal and approximal sites, respectively (p < 0.0001). Results from the three-month study confirmed this finding, with significant differences in plaque reduction (p < 0.0001) between the new and worn toothbrushes at 6 and 12 weeks for all sites. A significant difference (p < 0.0001) between the groups was also found for mean whole mouth gingivitis scores; this difference favoring the new brush at both 6 and 12 weeks. Examination of hard and soft oral tissues revealed no significant difference between the new and the worn brushes with respect to safety. It is concluded that a worn toothbrush is less effective than a new toothbrush for plaque removal and control of gingivitis.