Methylrosaniline Chloride (MRC) is recognized as a disinfectant, but recently is rarely used in the clinic, because of its cytotoxicity when used continuously with conventional concentrations (1% MRC). We have reported the antibacterial activity of MRC with lower concentration against Methicillin-resistant Staphylococcus aureus (MRSA). In this study, we evaluated the antifungal activity of MRC with lower concentrations. Antifungal activities of MRC against Candida spp. and Trichosporon spp. were tested. All strains tested were isolated from 106 blood or intravenous catheter samples at Juntendo University Hospital from 1995 to 2004. Minimum inhibitory concentrations against fungi were assayed by agar dilution, under both aerobic and anaerobic conditions. A 0.01% or less concentration of MRC solutions showed marked antifungal activity against Candida spp. and Trichosporon spp. under aerobic or anaerobic conditions. A 0.01% or less concentration of MRC should be reevaluated for the control of fungal infection and MRSA infection control.