Bacteria can be used for gene therapy via two strategies - either by transfection of eukaryotic host cells using bacteria (bactofection) or by alternative gene therapy that does not alter the host genome, but uses the prokaryotic expression system, which can be controlled or stopped from outside. While bactofection is optimal for gene substitution and DNA vaccination, alternative gene therapy is suitable for in situ delivery of proteins and treatment with intracellular bactochondria. A specific form of bacteria-mediated gene therapy is transkingdom RNA interference. In this review, advantages and issues related to bacterial vectors as well as the major applications in biomedical research are summarized. Despite numerous published experiments, especially in the treatment of solid tumors and gut infections, progress in the clinics lags behind and major improvements in the safety, and even more in the efficiency, of these approaches are needed.