A silkworm infection assay with the pathogenic fungus Rhizopus oryzae was established. Microbial culture broths were screened for anti-Rhizopus antibiotics using this assay. A new compound, tanzawaic acid R was isolated along with known and structurally related tanzawaic acids and arohynapene A from the culture broth of the hot spring-derived fungus Penicillium sp. BF-0005. The structure of tanzawaic acid R was elucidated by various spectroscopic data including 1D and 2D nuclear magnetic resonance spectroscopy. Tanzawaic acids A, B, C, and R and arohynapene A exhibited antifungal activity against R. oryzae. Tanzawaic acids A and B dose-dependently exerted therapeutic effects in the silkworm infection assay with R. oryzae.