Eleven strains of an entomopathogenic fungus, isolated and identified as Ophiocordyceps sobolifera, were screened for activity against 5 strains of Gram-negative and 5 strains of Gram-positive bacteria. Four of the isolates, Cod-KK1634, Cod-KK1643, Cod-KS1601, and Cod-SN1626, had activity against the test strains of Grampositive bacteria. Of these 4 extracts, the Cod-KK1643 extract had the lowest minimum inhibitory and bactericidal concentrations. The Cod-KK1643 extract exhibited both concentration- and time-dependent bactericidal activity. Moreover, the Cod-KK1643 extract induced morphological alterations in bacterial cells, including decreased cell size, a crushed appearance, and cell lysis. It is surprising to note that the extracts also inhibited MCF-7 cell proliferation, with a half-maximal inhibitory concentration of 47.09 ± 33.64 mg/mL after 72 hours. The extracts also inhibited MCF-7 cell migration, with the lowest percentage of relative closure achieved with Cod-KK1643. These findings represent what is to our knowledge the first information on the activities of O. sobolifera mycelial extracts against bacteria and the human breast cancer MCF-7 cell line.