Recent technological advances have enabled the production of split or feathered endings of individual toothbrush filaments. The purpose of this laboratory study was to compare a bi-level manual toothbrush containing standard end-rounded bristles with the same shaped toothbrush containing feathered filaments, 3 mm deep, on the outer raised bi-level rows to achieve subgingival access (SA) and remove artificial plaque at the gingival margin, or gingival margin cleaning (GMC). Simulated gingivae were prepared with a 0.2 mm space between the gingivae and tooth surface. The toothbrushes were aligned with the papillae at the gingival margin, and were tested using wet brushing conditions for 60 seconds at two strokes per second with a 15 mm stroke. Four toothbrushes of each type were evaluated four times, for a total of 16 assays per design. The SA maximum depth was recorded on pressure-sensitive paper under the simulated gingivae, and the length of gingival margin cleaning was recorded as the GMC efficacy. Readings were performed with 3x magnification by one investigator. In these laboratory assays, the bi-level toothbrush with feathered outer-row bristles significantly (p < 0.001) increased the ability of the bristles to achieve SA by 35.7% and GMC efficacy by 54.5% compared to an identical bi-level toothbrush with end-rounded bristles.