One of the biggest obstacles in cancer treatment is the development of chemoresistance. To overcome this, attempts have been made to screen novel anticancer substances derived from natural products. The purpose of this study is to find new anticancer candidates in the mycelium culture extract of mushrooms belonging to <i>Polyporus</i>. Here, we used a high-throughput screening to find agents capable of inhibiting cancer cell proliferation. The culture extract of <i>Polyporus Parvovarius</i> mycelium in DY medium (pp-DY) was effective. pp-DY inhibited cancer cell proliferation by inducing apoptosis and S-phase arrest. The anticancer property of pp-DY was not only effective against one type of cancer, but also against another type of cancer. Compound fractionation was performed, and the active ingredient exhibiting anticancer effects in pp-DY was identified as 3,4-dihydroxybenzaldehyde (Protocatechualdehyde, PCA). PCA, like pp-DY, inhibited the proliferation of cancer cells by inducing apoptosis and S-phase arrest. Furthermore, unlike conventional anticancer drugs, PCA did not increase the proportion of the side population that plays the most important role in the development of chemoresistance. Taken together, our data revealed the novel mycelium culture extract that exhibited anticancer property, and identified active ingredients that did not activate a proportion of the side population. These novel findings may have clinical applications in the treatment of cancer, particularly chemo-resistant cancer.