The genus Trichoderma includes significant agents for biological control. The mycelia extract obtained from the fermentation broth of Trichoderma harzianum was separated and the primary fractions were preliminary characterized using Thin Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FT-IR) and UV-visible Spectrophotometer (UV-vis). Consequently, we examined the spectral regions for characterization, assessed the interference of the prepared sample solutions regarding their resolution and evaluated the capacity to distinguish between Trichoderma harzianum. The active fraction is subjected to bioassay-guided purification through the thin-layer chromatography (TLC) technique following its extraction with five bioactive compounds. The genetic diversity observed was consistent with the findings from FTIR spectroscopy. The application of FTIR in the mid-infrared range of 449.75–3957.60 cm−1, using samples prepared with 50% alcohol facilitated the characterization and differentiation of Trichoderma harzianum based on their genetic diversity. The UV-vis spectrum exhibited distinct peaks, indicating the existence of secondary metabolites. From this study, methodology presents a rapid and efficient approach for the characterization and differentiation of Trichoderma samples. The TLC techniques are applicable to a variety of plant extracts and can also be utilized with different fungal species, whether (aerobic or anaerobic) as well as fungi can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.














