Pectobacterium and Dickeya species are the main causative agents for soft rot disease that adversely affect fruits and vegetables leading to considerable economic losses. Biological management with beneficial microorganisms is a promising alternative to hazardous bactericides. Therefore, the antagonistic activity of two different strains of Rahnella aquatilis was in vitro and in vivo evaluated against nine soft rotting bacterial strains. The antagonistic soil bacteria R. aquatilis strains 17 and 55 restricted the growth of nine soft rotting bacterial strains on nutrient agar plates, (7 Pectobaterium carotovorum strains and 2 Dickeya chrysanthmi strains). Transmission electron micrographs of P. carotovorum Pep3B cells antagonized with R. aquatilis strain 17, showed damaged cells with disrupted plasma membrane releasing the cellular contents. To examine whether R. aquatilis 17 could be an effective biological control agent for pepper soft rot disease, two applications were conducted. The pepper seedlings were pretreated, before the pathogen, with R. aquatilis 17 through leaves and roots. All seedlings pretreated with the antagonistic strain 17 showed reduced susceptibility towards the P. carotovorum Pep3B, increased fresh, dry weights and seedlings' height relative to controls. R. aquatilis 17 inoculation has positively influenced the physiological parameters evaluated, such as chlorophyll content, carotenoids, phenolics, flavonoids, protein concentration as well as proline concentration. The obtained results revealed that R. aquatilis 17 mitigated the effect of P. carotovorum on pepper seedlings and promoted their growth, which means that it has a high probability of being an effective biological control agent and a plant-promoting bacterium.