Bio-sulfur can be produced in the process of desulfurization from a landfill and collected by some microorganism such as Thiobacillus sp. as a sulfur element. In order to investigate practical use of bio-sulfur as an agent for controlling plant disease, in vitro antifungal activity of bio-sulfur was tested against Colletotrichum orbiculare known to cause cucumber anthracnose. Efficacy of bio-sulfur for suppressing anthracnose disease was also evaluated in vivo using cucumber leaves. Mycelial growth of C. orbiculare on medium containing bio-sulfur was inhibited. Disease severity of cucumber leaves pre-treated with bio-sulfur was significantly decreased compared to that of untreated ones. To illustrate how bio-sulfur could suppress anthracnose disease, structures of cucumber leaves infected with C. orbiculare were observed under a fluorescent microscope and a scanning electron microscope (SEM). Cucumber leaves pre-treated with bio-sulfur showed a low rate of appressorium formation whereas untreated ones showed abundant appressoria. Shrunk fungal hyphae were mostly observed on bio-sulfur-pretreated leaves by SEM. Similar results were observed on leaves pre-treated with a commercial fungicide Benomyl®. These results suggest that inhibition of appressorium formation of C. orbiculare by bio-sulfur may contribute to its suppression of cucumber anthracnose.