The effects of seedlings colonized PGPR stains on tomato bacterial wilt disease suppression and yield were investigated via continuously 3 season field experiments in this study for providing a new strategy to maintain the tomato sustainable production. Compared with the control (BIO) transplanted with common seedlings cultivated in ordinary nursery substrate, seedlings colonized PGPR stain produced by bio-nursery substrate were transplanted in the field amended with bio-organic fertilizer (BIONS) to evaluate the tomato yield enhancement and bacterial wilt disease suppression ability as well as its influence on microbial biomass and soil properties in a plastic greenhouse. Results showed that compared to BIO, BIONS significantly increased tomato yield by 38.86%, 47.87% and 34.60% in the first, second and third seasons, respectively. Meanwhile, tomato bacterial wilt disease incidence of BIONS was prominently lower than BIO. Moreover, BIONS application significantly increased rhizosphere bacteria but decreased fungi populations. During the three seasons, the contents of ammonium nitrogen (NO₃^--N) and nitrate nitrogen (NH₄⁺-N) were higher in BIONS than in BIO; ammonium nitrogen amount had a positive correlation with tomato yield; and negative correlation was observed between disease incidence and the contents of ammonium nitrogen and nitrate nitrogen. The above results prove that continuous application of the bio-nursery substrates in greenhouse can effectively control tomato bacterial wilt disease, thus can increase tomato yields.