The antibacterial activity of potential retrograde root filling materials was assessed using the agar diffusion inhibitory test. A light-cured glass ionomer cement (Vitrebond) and three reinforced zinc oxide-eugenol cements (Kalzinol, IRM & ethoxybenzoic acid [EBA] cement) were compared with amalgam, a commonly used retrograde root filling material. Forty standardized pellets of each material were produced. Fresh, and materials aged for 1 week in sterile distilled water, were placed on blood agar plates inoculated with Streptococcus anginosus (milleri) or Enterococcus faecalis. At intervals of 3, 7 and 10 days, the presence and diameter of zones of inhibition were recorded. The diameter of the zones of inhibition increased with time for all materials, both fresh and aged. Vitrebond had the most pronounced antibacterial activity against both bacteria, producing the largest zone of inhibition, followed by Kalzinol. Amalgam had no measurable inhibitory effect whether aged or fresh, regardless of the period of exposure. There was no statistically significant difference in the response of the two bacteria. However, there were statistically significant differences between materials, period of exposure, and between fresh and aged materials (P < 0.001). Kalzinol, IRM and EBA cement were more antibacterial when aged than fresh, whilst Vitrebond was more active when fresh. Vitrebond was the only material for which the diameter of the zones of inhibition was reduced after ageing but it had the most pronounced antibacterial activity compared with the other materials. The antibacterial activity of the materials was ranked in the following order: Vitrebond > Kalzinol > (IRM = EBA cement) > amalgam.