Investigation of the properties and mechanisms of the interactions of root-colonizing biocontrol bacteria and plant pathogens is necessary to optimize the biocontrol strategies. In the present study, the interaction of a biocontrol strain Bacillus amyloliquefaciens T-5 tagged with a green fluorescent protein marker and a bacterial wilt pathogen Ralstonia solanacearum QL-Rs1115 tagged with red fluorescent protein marker was studied on tomato roots using different inoculation methods. The results showed that in the co-culture experiment, the population of pathogen QL-RFP was decreased by increasing the initial inoculum concentration of biocontrol strain. In the greenhouse experiment, both strains T-5-GFP and QL-RFP colonized tomato roots (root tips, root hairs, primary roots, and root junctions) and formed a biofilm on the root surfaces as determined by dilution plating and confocal laser scanning microscopy (CLSM) techniques. However, the root colonization of pathogen strain QL-RFP was almost completely suppressed in the presence of biocontrol strain T-5-GFP when both soil and plant seedlings were treated with T-5-GFP. The results of this study revealed the effectiveness of strain B. amyloliquefaciens T-5 as a biocontrol agent against tomato wilt pathogen and the significance of inoculation method used to inoculate biocontrol strain.