The study aimed to evaluate intracanal irrigation procedures in eradicating bacteria from surface, shallow and deep layers of root dentine using extracted human teeth. Artificial bacterial smear layer was successfully produced by rubbing a mixture of dental plaque and artificially decalcified dentine or carious dentine on root canal walls. The reservoir holes were 3.5 mm in depth, 1 mm in diameter prepared 1.5 mm apart and parallel to the root canals on the decrowned planes, in which five separate bacterial species were placed (Actinomyces israelii, Fusobacterium nucleatum, Propionibacterium acnes, Streptococcus mutans and Streptococcus sanguis). Bacterial eradication after irrigation of the prepared canals was determined by bacterial recovery (i) from the root canal surfaces and shallow layers where bacteria were smeared artificially and (ii) from deeper layers of root canal dentine reservoir holes. Ultrasonic irrigation with 5.5% and 12% NaOCl eradicated bacteria from artificial smear layer (P < 0.0001), whilst 12% NaOCl irrigation with a syringe was insufficient. Ultrasonic irrigation with water or 15% EDTA-failed to eradicate bacteria from smeared surfaces. Ultrasonic irrigation with 12% NaOCl killed A. israelii, F. nucleatum, P. acnes, S. mutans, and S. sanguis placed in reservoir channels, although for F. nucleatum, a very small number of bacteria remained in five samples out of 12. Ultrasonic irrigation with less concentrated NaOCl failed to eliminate bacteria completely from reservoir channels in most samples. Ultrasonic irrigation with 12% NaOCl appeared to eliminate bacteria efficiently from surface, shallow and deep layers of root dentine.