The aim of this study was to investigate the antimicrobial activity of propolis-based intracanal medicament against Enterococcus faecalis using infected dentine models, and to compare its antimicrobial efficacy with that of the non-setting calcium hydroxide paste when used as a short-term medication for 1 and 2 days. A total of 50 dentine discs of 7-mm length was obtained from extracted human teeth. Five dentine discs were kept sterile to serve as a negative control. The remaining 45 were contaminated with E. faecalis and divided into two groups (n = 20) in addition to five discs that served as a positive control. The discs were treated as follow: 20 discs were filled with propolis, while the other 20 discs were filled with non-setting calcium hydroxide. Microbiological sampling was performed utilising sterile paper point, headstrom file and disc immersion. Results showed that propolis was significantly more effective than non-setting calcium hydroxide against E. faecalis after short-term application, which made comparison from this prospect unlevelled. The most effective microbiological sampling technique was abrading the lumen with headstrom file. Propolis is very effective as intracanal medicament in rapidly eliminating E. faecalis ex vivo.