The growth-inhibiting activities of materials derived from the fruiting body of Cordyceps militaris cultured on Bombyx mori pupae (CM-1) and pupae separated from the culture (CM-2), and the fruiting body of Paecilomyces japonica cultured on B. mori pupae (PJ-1) and pupae separated from the culture (PJ-2), fresh B. mori larvae (BML), fresh B. mori female pupae (BMP), and Morus alba leaves (MAL) towards eight lactic acid-producing bacteria and 11 harmful intestinal bacteria were examined using an impregnated paper disc bioassay. At 10 mg per disc, methanolic extracts from CM-1 and CM-2 strongly inhibited growth of Clostridium difficile ATCC 9689, C. paraputrificum ATCC 25780, and C. perfringens ATCC 13124 without adverse effects on the growth of eight lactic acid-producing bacteria and the other eight harmful bacteria. The methanolic extracts from PJ-1, PJ-2, BML, BMP, and MAL did not affect growth of all test bacteria. The growth-inhibiting principle of CM-1 and CM-2 towards test clostridia was characterized as cordycepin by spectroscopic analysis. The contents of cordycepin in dried CM-1 and CM-2 were 0.69% and 0.54%, respectively. These results suggest that cordycepin may be produced from the fruiting body of C. militaris cultured on B. mori pupae and then translocated to its host insect and accumulated. Structure-growth inhibition relationships of cordycepin and its eight derivatives against C. perfringens ATCC 13124 indicate that a deoxy form at either 3 or 2 position appears critical for the inhibitory activity. Natural cordycepin and its two analogues, 2 -deoxyadenosine and tubercidin, merit further study as a potential antibacterial agent against various diseases caused by harmful intestinal bacteria such as clostridia.