Aim: To compare in vitro the antimicrobial effect of acidic and alkaline intra-canalmedicaments on two resistant forms of microorganisms.Methodology: Part I, forty eight agar plates were swabbed with the prepared microbialsuspensions containing either Enterococcus faecalis or Candida albicans. Then the testedmedicaments (10% calcium hydroxide, 10% citric acid, 5% gluconic acid and saline as a control)were pipetted onto sterile filter paper discs placed on the agar plates. The plates were incubated.Then, the diameters of inhibition zones were recorded for data analysis. Part II: Eighty extractedsingle rooted human teeth were selected. Following teeth standardization, instrumentation andsterilization, the root canals were equally divided into 2 groups either contaminated withEnterococcus faecalis or Candida albicans. Each group was equally divided and filled with oneof the tested intra-canal dressing materials. After incubation, microbiological samples werecollected with sterile paper points, plated on agar plates and the number of colony forming unitswas determined.Results: For the agar diffusion test, citric acid and calcium hydroxide showed highestinhibition zone against both microorganisms. While in part II of this study, there was nostatistical significant difference between calcium hydroxide, citric acid and gluconic acid. Yetthese groups showed statistical significant difference with saline group. Comparing the two acidsused, citric acid was more effective against Enterococcus faecalis, while gluconic acid was moreeffective against Candida albicans.Conclusion: The different tested materials used in this study exerted differentantimicrobial effect against Enterococcus faecalis and Candida albicans.