The purpose of this in vitro study was to evaluate the antibacterial activity of five different root-canal sealers (RoekoSeal, Ketac-Endo, AH Plus, Sealapex, Sultan). With the use of Enterococcus faecalis as a test organism, both the agar-diffusion test (ADT) and direct-contact test (DCT) were performed. For DCT, sealers were mixed and placed on the sidewall of microtiter plate wells. A 10-μl bacterial suspension was placed on the tested material samples. Bacteria were allowed to directly contact to the sealers for 1 h at 37°C. Bacterial growth was then spectrophotometrically measured through every 30 min for 19 h by using an Anthos Labtec HT 2. For ADT, a 200-μl bacterial suspension was spread on brain-heart infusion agar plates. Freshly mixed sealers were poured into uniform wells punched in the agar. After periods of incubation at 37°C for 24 h and 7 days in humid atmosphere, the zones of inhibition of bacterial growth on agar plates were observed and measured. Ketac-Endo, Sultan, and AH Plus had similar results for DCT. These sealers were more potent bacterial-growth inhibitors than Sealapex and RoekoSeal. According to ADT, RoekoSeal showed no antibacterial activity. There was no significant difference among AH Plus, Sealapex, and Sultan (p > 0.05). Ketac-Endo demonstrated lower antimicrobial activity than these sealers (p < 0.05). Time had no effect on the antibacterial activity of the tested sealers (p > 0.05). The antibacterial efficiency of the materials varied according to the tests used. It was concluded that the technique, time, and ingredients of the tested material can affect the results of the microbiological studies.