Populations of pathogenic Ralstonia solanacearum and functional bacteria groups in the rhizospheric soils of continuous cropping tomato and pepper applied with a kind of bio-organic fertilizer (BOF) and with conventional fertilizer (CK) were studied using the real-time quantitative PCR techniques. As compared with CK, BOF increased the yields of tomato and pepper by 26.0% and 19.9%, respectively, and decreased the incidence of bacterial wilt on tomato and pepper by 41.5% and 44.7%, respectively. BOF increased the population of Azotobacter and Pseudomonas flurosecens by 23.5% and 29.5%, and by 25.8% and 20.2%, and decreased the population of R. solanacearum by 73.2% and by 90.1%, respectively, in the rhizospheric soil of healthy tomato and pepper. In conclusion, the bio-organic fertilizer is capable of regulating the composition of microflora in rhizosphere soil of crops, reducing the incidence of soil-borne plant disease, and promoting plant growth. Real time PCR techniques are quick and accurate in determination of the population of functional microbes in soil.