Fusarium oxysporum, a major soil-borne fungal pathogen, causes vascular wilt, damping-off, and root rot diseases on over 100 cultivated plant species. Mechanisms of root colonization by F. oxysporum in Arabidopsis thaliana were studied through in planta 3-dimensional time-lapse documentation using confocal and multi-photon microscopy. Data from individual encounter sites were acquired repeatedly over a several day period without physical manipulation or retrieval from the growth chamber. In vivo observations were facilitated by transformation of F. oxysporum for constitutive cytoplasmic expression of the fluorescent protein ZsGreen, and host responses were monitored using autofluorescence or GFP-tagged endoplasmic reticulum. Penetration into the vascular system occurred primarily in the meristematic region of primary and lateral roots. Fungal hyphae may release phytotoxin(s) that compromise host cells not directly in contact with hyphae. This novel approach was essential for visualizing the dynamic interactions between F. oxysporum and A. thaliana from both the host and pathogen sides.