This study examined the phytochemical profile and the in vitro anti-proliferative effects of a hot water mycelial extract from the edible mushroom Pleurotus sp. on NB4 human leukemia cells. Flow-cytometry analyses were used to measure cell viability, cell cycle, and apoptosis in cells incubated 24 h with the extract at doses of 100 and 200 µg/mL. Pleurotus sp. extract reduced cell viability, particularly at the concentration of 200 µg/mL to 82% compared to control cells, and induced apoptosis demonstrated by an increase in the number of annexin V-FITC+ cells (25% at 200 µg/mL). The NB4 cells were arrested in the G2/M phase thus supporting a cell-cycle dependent anticancer mechanism. Although carbohydrates (76.8%, w/w) appear to be the most important antitumor compound, secondary metabolites-like phenolics would also contribute to the anti-proliferative activity. The results indicate that Pleurotus sp. mycelia obtained by submerged fermentation may be an interesting renewable resource for developing functional foods and new antitumor therapeutic agents.