The effectiveness of sodium hypochlorite (NaOCl) (2.5%) or iodine (10%) for decontamination of the operation field (tooth, rubber dam, and gasket [Oraseal]) was compared by using bacterial cultivation. In addition, the final samples were also assessed for bacteria by using polymerase chain reaction. Teeth (n = 63) receiving root canal treatment were polished with pumice, isolated with rubber dam, and their margins sealed with Oraseal. The operation field was disinfected with hydrogen peroxide (30%), followed by iodine (n = 31) or NaOCl (n = 32), before and after access cavity preparation. The operation field was sampled before and after each decontamination, giving four samples per field. After the final decontamination, there was no significant difference (p = 0.602, 0.113, 0.204) in recovery of cultivable bacteria from various sites in either group. However, bacterial DNA could be detected significantly (p = 0.010) more frequently from the tooth surfaces after iodine (45%) compared with NaOCl (13%) decontamination, although on the rubber dam or Oraseal surfaces there was no difference. Root canal sampling for polymerase chain reaction might be better preceded by NaOCl decontamination than by iodine, based on the findings.