The retention and survival of microorganisms on toothbrushes pose a threat of recontamination for certain patients at risk. In order to measure the influence of brush design and optimize the choice of toothbrush model for complementary studies, the in vitro retention of three microbial species (Porphyromonas gingivalis ATCC 33277, Streptococcus mutans ATCC 25175 and Candida albicans ATCC 26555) was evaluated for three types of toothbrush. Two series of standardized experiments were carried out for each brush and microorganism. The first series tested the retention of the microorganisms on the head portion of the brush, while the second measured retention on the head of the brush and the part of the handle inserted in the mouth during brushing. For each series, the microorganisms were counted at T0 and T24 (after storage of the brushes at room temperature for 24 h). Depending on the microorganism studied, from 0.2% to 2% of the initial inoculum was retained on the brush. The number detected increased with the size of the exposed area. After 24 h, P. gingivalis and S. mutans were found on only one type of brush. C. albicans survived on all three. These results confirm that microorganisms can quickly colonize toothbrushes.