Abstract Banana ( Musa acuminata ) growth for commercial purposes requires high amounts of chemical fertilizers, generating high costs and deleterious effects on the environment. In a previous study, we demonstrated that two plant growth-promoting rhizobacteria (PGPR), Bacillus amyloliquefaciens Bs006 and Pseudomonas palleroniana Ps006, isolated in Colombia, could partially replace chemical fertilizers for banana seedling growth. In a second work, the effects of the two inoculants on banana transcripts were found to occur at different times, earlier for Bs006 and later for Ps006. This leads to the hypothesis that the two rhizobacteria have different colonization dynamics. Accordingly, the aim of this work was to analyze the dynamics of root colonization of the two PGPR, Bs006 and Ps006, on banana growth over a time frame of 30 days. We used fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM), followed by three-dimensional reconstruction and quantitative image analysis. Bacillus amyloliquefaciens Bs006 abundantly colonized banana roots earlier (from 1 to 48 h), ectophytically on the rhizoplane, and then decreased. Pseudomonas palleroniana Ps006 was initially scarce, but after 96 h it increased dramatically and became clearly endophytic. Here we identify and discuss the potential genetic factors responsible for this complementary behavior. This information is crucial for optimizing the formulation of an effective biofertilizer for banana and its inoculation strategy.