A benomyl-tolerant strain (94110Tr) of Trichoderma spp. was isolated from airborne fungi in a growing room for sawdust-based cultivation of Lentinula edodes (Berk.) Pegler in Hokkaido. The strain was identified as T. harzianum Rifai, so the antifungal activity of benomyl (BEN), thiabendazole (TBZ), benzalkonium chloride (BAC), chlorhexidine digluconate (CHG), and glutaraldehyde (GLA) were tested against six strains of T. harzianum, including 94110Tr. It was found that 94110Tr was more resistant than the others not only to BEN but also to TBZ, but it had no explicit tolerance to BAC, CHG, or GLA, similar to the other strains. CHG and GLA showed potent antifungal activity against most of the six strains. It is estimated that CHG and GLA are available for disinfection of the benomyl-tolerant strains of T. harzianum found in edible-mushroom factories.