Background: There have been many reports on microbial contamination of antiseptics and disinfectant used in the hospitals. Mid to low level disinfectants such as chlorhexidine gluconate, quaternary ammonium, phenols and benzalkonium are reported to be the one with high risk of microbial contamination. We investigated microbial contamination of 0.05% chlorhexidine gluconate solution used in our hospital. Methods: 0,05% chlorhexidine gluconate solution and 0.05% chlorhexidine gluconate cotton balls used in the general ward and intensive care unit were randomly collected for microbial culture. Also, sterile water, 20% chlorhexidine gluconate and staining solution, which is mixed in the preparation room to make 0,05% chlorhexidine gluconate solutions, were collected for bacterial culture to evaluate the focus of microbial contamination. Results: Total of 31 chlorhexidine gluconate samples was randomly collected from general wards. intensive care unit and preparation room for microbial culture. Seven of thirteen 0.05% chlorhexidine gluconate solution and 5 of twelve 0.05% chlorhexidine soaked cotton balls kept in a canister were contaminated by Burkholderia cepacia. Sterile water used for dilution of 20% chlorhexidine gluconate to make 0.05% chlorhexidine and originally purchased 20% chlorhexidine gluconate were not contaminated by microorganism. But staining solution that is used as an additive to differentiate from other diluted disinfectants was contaminated by Bukholderia cepacia. 0.05% chlorhexidine gluconate solutions that were mixed in the preparation room were also contaminated by same organism. Source of contamination was thought to be staining solution. All the previously made chlorhexidine. solutions and staining solution were discarded. Staining solution was not used afterward. Several processes in the handling of chlorthexidine solution were corrected and no microbial contamination was found afterward. Conclusion: Diluted chlorthexidine gluconate solutions are always under a risk of microbial contamination if any of the process in the handling is overlooked. Staining solution as an additive to disinfectants should be used carefully.