Aim: The aim of this study was the monitoring of different mechanisms involved in the antibacterial activity of the biocontrol agent, Stenotrophomonas maltophilia (PD4560), against Ralstonia solanacearum in vitro and in vivo. Optimization of conditions that favour these mechanisms was the second target of this study. Methods and Results: Proteolytic activity of Sten. maltophilia (PD 4560), was tested on skimmed milk medium. The biocontrol agent was able to produce an alkaline serine protease enzyme with a molecular weight of 40 KDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. Spraying of salicylic acid (SA) led to an increase in the efficacy of Sten. maltophilia in controlling the Ralstonia potato wilt while spraying of ammonium sulphate (AmS) did not affect the biocontrol efficacy. The efficacy was correlated with the expression of protease enzyme genes; Prt genes (mainly PrtP and Prt4) and PR genes (mainly PR-1 and PRQ) as evaluated using real-time polymerase chain reaction analysis. Conclusions: The biocontrol activity of Sten. maltophilia can be attributed to the direct mechanism alkaline serine proteolytic enzyme production and through induction of host systemic acquired resistance as indirect mechanism. Tuber bulking was the most suitable physiological growth stage to apply either SA or the biocontrol agent. Significance and Impact of the Study: Both SA and peat-moss as an organic carrier enhanced the antibacterial efficiency of the biocontrol agent. Application of Sten. maltophilia is more suitable under alkaline soil conditions.