The wiping of high-touch healthcare surfaces made of metals, ceramics and plastics to remove bacteria is an accepted tool in combatting the transmission of healthcare-associated infections (HCAIs). In practice, surfaces may be repeatedly wiped using a single wipe, and the potential for recontamination may be affected by various factors. Accordingly, we studied how the surface to be wiped, the type of fibre in the wipe and how the presence of liquid biocide affected the degree of recontamination. Experiments were conducted using metal, ceramic and plastic healthcare surfaces, and two different wipe compositions (hygroscopic and hydrophilic), with and without liquid biocide. Despite initially high removal efficiencies of >70% during initial wiping, all healthcare surfaces were recontaminated with E. coli, S. aureus and E. faecalis when wiped more than once using the same wipe. Recontamination occurred regardless of the fibre composition of the wipe or the presence of a liquid biocide. The extent of recontamination by E. coli, S. aureus and E. faecalis bacteria also increased when metal healthcare surfaces possessed a higher microscale roughness (<1 µm), as determined by Atomic Force Microscopy (AFM). The high propensity for healthcare surfaces to be re-contaminated following initial wiping suggests that a “One wipe, One surface, One direction, Dispose” policy should be implemented and rigorously enforced.