Antagonistic actinomycete strains isolated from the environment are valuable tools for an eco-friendly, healthy, and safe control of phytopathogenic fungi. We have evaluated the culture filtrate of Streptomyces griseorubens E44G, an actinomycete strain isolated from soil, on the growth and ultrastructure of hyphal cells of the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici, the causal agent of Fusarium wilt disease of tomato. The effect of the Streptomyces culture filtrate on some of the carbohydrate fractions in the hyphal cell of the pathogen using gold-labeled lectin complexes was also elucidated. Of the concentrations of S. griseorubens E44G culture filtrate tested, the highest (400 μL) had the most potent antifungal effect on the mycelial growth of the fungus. At this concentration, some changes in the morphology of the fungal hyphae were observed by scanning electron microscopy, and a number of dramatic changes in the ultrastructure of the hyphal cells of the fungus were observed by transmission electron microscopy. Ultracytochemical localization of carbohydrate fractions of the hyphal cell of the fungus revealed the presence of a very high quantity of chitin in the cell wall which was digested following exposure to the culture filtrate of S. griseorubens E44G, indicating the presence of a chitinase enzyme in that filtrate. The ultracytochemical investigations also indicated the presence of mannose, glucose, and galactose in the fungal cell wall, as well as the absence of glucosides. Moreover, the fungal cell cytoplasm contained glucosides and galactose but not chitin. These results confirm that the chitinase enzyme was produced by S. griseorubens E44G and that this enzyme may play a role in the potential of this strain as an antifungal agent against F. oxysporum f. sp. lycopersici.