Pseudomonas fluorescens B16 has been shown to be a promising biological control agent for the promotion of plant growth, root colonization, and suppression of bacterial wilt. This study investigated the dispersion of B16 from seed to radicle, and to the whole root system, and illustrated population changes within cucumber seeds and radicles using scanning electron microscopy (SEM), fluorescence microscopy (FM) and immunofluorescence microscopy (IFM). When cucumber seeds were soaked in a bacterial suspension of B16, bacterial cells entered the seeds through the pore at the base, colonized the inner plane, and proliferated germination advanced. Population densities of B16 were gradually increased, and increased initial populations within newly growing roots. The root surface was covered by cylindrical rod-shaped bacterial cells, and junctions between epidermal cells were covered preferentially. Previous studies exploring root colonization of rhizobacteria have not focused on colonized populations inside seeds. Our microscopic observations confirmed the importance of bacterial cells colonized inside seed coats. Population densities of B16 were then naturally sustained up to harvesting time. The results obtained in this study provide about a novel insight into the commercial application of biological control agents within seeds.