RNA interference (RNAi) represents a powerful mechanism for the knockdown of any target gene of interest. It is based on the action of small double-stranded RNA molecules (siRNAs) which are complementary to the target sequence. Crucial for the induction of RNAi, particularly for any therapeutic in vivo applications, is the delivery of siRNAs or small hairpin RNA (shRNA)-encoding DNA constructs. Extending the portfolio of viral or non-viral strategies, bacteria have been shown to be able to deliver shRNA-encoding DNA into tumor cells. In a process termed 'transkingdom RNA interference' (tkRNAi), the genetically modified bacteria themselves produce shRNAs which, upon bacterial infection, become available for the target cells for further processing and induction of RNAi. In 'bacteria-mediated RNAi' (bm-RNAi), the bacteria only deliver the shRNA-encoding DNA construct with subsequent transcription of the shRNA in the target cells. This review briefly discusses general aspects of the RNAi mechanism and various delivery systems, and then describes tkRNAi and bm-RNAi in the context of therapeutic applications.