Roridin E, a constituent of Podostroma cornu-damae, has excellent medicinal properties and exhibits powerful anticancer effects. However, being a poisonous mushroom, P. cornu-damae is difficult to find in its natural environment. Therefore, only a limited amount of roridin E is isolated from P. cornu-damae. We isolated roridin E from mycelial culture medium of P. cornu-damae and examined its effects on five breast cancer cell lines. The purity of the isolated roridin E was 96%. It exhibited anticancer effects on all five breast cancer cell lines, with an IC50 of at least 0.0006 mg/L. To the best of our knowledge, this is the first report on the investigation of effects of roridin E isolated from the mycelium of P. cornu-damae mushrooms on five breast cancer cell lines. However, safety issues should be investigated and addressed in future studies before roridin E is used in clinical settings.