The detailed mycochemical analysis of the <i>n</i>-hexane extract of <i>Pholiota populnea</i> led to the isolation of four new lanostane diesters, named pholiols A-D (<b>1</b>-<b>4</b>), together with an acyclic triterpene, (3<i>S</i>,6<i>E</i>,10<i>E</i>,14<i>E</i>,18<i>E</i>,22<i>S</i>)-2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (<b>5</b>), ergosterol (<b>6</b>), and 3β-hydroxyergosta-7,22-diene (<b>7</b>). The isolation was carried out by multistep flash chromatography, and the structures were elucidated using extensive spectroscopic analyses, including 1D and 2D NMR and MS measurements. The isolated metabolites (<b>1</b>-<b>6</b>) were investigated for cytotoxic activity against Colo205 and Colo320 colon adenocarcinoma and nontumoral MRC-5 cell lines. Among the tested compounds, ergosterol (<b>6</b>) showed substantial cytotoxic activity against all cell lines with IC<sub>50</sub> values of 4.9 μM (Colo 205), 6.5 μM (Colo 320), and 0.50 μM (MRC) with no tumor cell selectivity. A P-glycoprotein efflux pump modulatory test on resistant Colo320 cells revealed that pholiols A (<b>1</b>) and B (<b>2</b>) and linear triterpene polyol <b>5</b> have the capacity to inhibit the efflux-pump overexpressed in the cells. Moreover, the drug interactions of triterpenes with doxorubicin were studied by the checkerboard method on Colo 320 cells. Pholiols B (<b>2</b>) and D (<b>4</b>) interacted in synergistic and acyclic triterpene <b>5</b> in a very strong synergistic manner; the combination index (CI) values at 50% of the growth inhibition dose (ED<sub>50</sub>) were found to be 0.348, 0.660, and 0.082, respectively. Our results indicate that <i>P. populnea</i> is a promising source for finding new triterpenes with significant chemosensitizing activity on cancer cells.