The aim of this study was to investigate bacterial survival and biofilm formation on toothbrushes. Fifteen healthy volunteers each used a normal toothbrush and an antibacterial toothbrush of the same design for two separate 5 week periods. Bacteria were removed from the brush head by swabbing and mechanical agitation in 10ml of tryptone soya broth, cultured aerobically on selective and non-selective media, and classified by Gram staining, catalase and oxidase tests. Survival of Staphylococcus epidermidis and Pseudomonas aeruginosa was monitored in the laboratory on both types of brush over 8 days. Scanning electron microscopy was used to observe biofilm formation on antibacterial and conventional brushes used for various times. Numbers of bacteria isolated from conventional and antibacterial brushes from different individuals ranged from 8.3×103 to 4.7×106 and from 1×102 to 1.2×106 colony-forming units/ml, respectively. A larger number of bacteria were isolated from conventional brushes than from antibacterial brushes used by the same individuals but no statistically significant difference was demonstrated. No differences in the relative proportions of Gram-negative and Gram-positive rods or cocci were seen. Staphylococci, presumptive coliforms and pseudomonads were isolated from 48%, 28% and 16% of brushes, respectively. Pseudomonas aeruginosa was viable for at least 4 days on conventional, and 2–3 days on antibacterial, brushes, whilst S. epidermidis survived for 6–8 days on antibacterial and more than 8 days on conventional brushes. Biofilms formed on the heads and bristles of both conventional and antibacterial brushes. Extensive, mixed community biofilms developed after several months of use. We conclude that toothbrushes may be a reservoir of opportunistic pathogens including staphylococci and pseudomonad-like organisms and must be considered as a potential source of haematogenous infections and cross-infection.