Fusarium wilt is caused by the soil‐inhabiting fungus Fusarium oxysporum ff. spp. and is one of the most devastating plant diseases, resulting in losses and decreasing the quality and safety of agricultural crops. We recently reported the structures and biochemical properties of two biotin‐binding proteins, streptavidin C1 and C2 (isolated from Streptomyces cinnamonensis strain KPP02129). In the present study, the potential of the biotin‐binding proteins as antifungal agent for Fusarium wilt pathogens was investigated using recombinant streptavidin C1 and C2. The minimum inhibitory concentration of streptavidin C2 was found to be 16 µg ml–1 for inhibiting the mycelial growth of F. oxysporum f.sp. cucumerinum and F. oxysporum f.sp. lycopersici, while that of streptavidin C1 was found to be 64 µg ml–1. Compared with the nontreated control soil, the population density of F. oxysporum f.sp. lycopersici in the soil was reduced to 49·5% and 39·6% on treatment with streptavidin C1 (500 µg ml–1) and C2 (500 µg ml–1), respectively. A greenhouse experiment revealed that Fusarium wilt of tomato plants was completely inhibited on soil drenching using a 50‐ml culture filtrate of the streptavidin‐producing strain KPP02129.