The aims of this study were to determine the microbial contamination of gutta-percha and resilon cones and to evaluate their antimicrobial activities against five different microorganisms (Staphylococcus aureus ATCC 29213, Pseudomonas auroginosa ATCC 27853, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212 and Candida albicans ATCC 10231). Resilon and gutta-percha cones from seven manufacturers were used in this study. Antimicrobial activities of the cones against five different microorganisms were tested by agar disc diffusion method. To detect the bacterial contamination of cones during clinical use, the cones taken from the previously opened packages were placed into tripticase soy agar. Bacterial growth was detected in 3 out of 28 packages of cones. Clinical use of all the cones led to increase in bacterial contamination. ProTaper (R) and Mtwo (R) gutta-percha cones were determined to have lowest antibacterial activities against S. aureus and P. aeruginosa strains. The cones may be contaminated right after opening their packages and/or during clinical use. It is recommended that they should be disinfected before placing into the canals.