Encapsulation of biological material in the permiselective membrane allows to construct a system separating cells from their products, which may find biotechnological as well as biomedical applications in biological processes regulation. Application of a permiselective membrane allows avoiding an attack of the implanted microorganisms on the host. Our aim was to evaluate the performance of Bacillus subtilis encapsulated in an elaborate membrane system producing listeriolysin O, a cytolysin from Listeria monocytogenes, with chosen eukaryotic cells for future application in anticancer treatment. The system of encapsulating in membrane live Bacillus subtilis BR1-S secreting listeriolysin O was proven to exert the effective cytotoxic activity on eukaryotic cells. Interestingly, listeriolysin O showed selective cytotoxic activity on eukaryotic cells: more human leukemia Jurkat T cells were killed than human chronic lymphocytic B cells leukemia at similar conditions in vitro. This system of encapsulated B. subtilis, continuously releasing bacterial products, may affect selectively different types of cells and may have future application in local anticancer treatment.