Fusarium proliferatum is a broad host range, mycotoxin-producing, seed-borne pathogen of maize. A green fluorescent, hygromycin-resistant F. proliferatum transformant (Fp-70–2-5-G2) was developed to provide markers to track movement and colonization of bait organic matter in non-sterile soil microcosms. Maize seeds that were heat-killed and re-colonized with Fp-70–2-5-G2 served as sources of inoculum. They were added to non-sterile soil together with heat-killed, non-colonized maize seeds (baits), at several temperatures, soil matric potentials, and source-to-bait distance combinations. Baits were retrieved from soil and plated onto Nash-Snyder medium amended with 1 µg mL−1 hygromycin. Fp-70–2-5-G2 grew through non-sterile sieved soil and colonized maize seed; distance, temperature and soil matric potential had significant effects (P < 0.0001, P = 0.0365 and P < 0.0001, respectively) on growth and colonization. The maximum distance that Fp-70–2-5-G2 grew and colonized maize seed was 1.5 cm at 25°C and −0.005 MPa matric potential. The maximum colonization of baits by Fp-70–2-5-G2 occurred at 0 cm (80%) and 0.5 cm (50%) source-to-bait distance at 25°C and −0.005 MPa matric potential. Growth through soil and colonization of bait organic matter was significantly less in soil with an intact field structure. In addition to plant and seed colonization, F. proliferatum may have an active soil inhabitant phase in its life cycle.