Plant pathogenic fungi are the main causative agents of diseases associated with significant productivity and economic losses in agriculture. Thus, the rapid and early identification of plant pathogens is essential for directing effective management and control strategies for diseases to prevent or reduce their severity. The objective of this study was to characterize and discriminate different species of plant pathogenic fungi using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and chemometric analysis. Five fungal species of the genera Colletotrichum, Fusarium, Macrophomina, Neoscytalidium, and Pseudofusicoccum, isolated from agricultural crops, fruit trees, and ornamental plants, were used. Discrimination of the fungal samples was conducted using different fingerprint regions of ATR-FTIR spectra and partial least squares-discriminant analysis (PLS-DA). The results showed that regions I (800–1800 cm−1) and III (800–1800 and 2800–2990 cm−1) can be used as spectroscopic biomarkers for the differentiation and classification of fungi by ATR-FTIR, as PLS-DA revealed adjustment quality and satisfactory prediction, generating values of R2 = 0.96 and Q2 = 0.60 in cross-validation. The most relevant infrared absorption bands in the separation of classes were the C–O stretching vibrations, bending of C-H bonds, amide III bands, and C–C stretching. This method can be an important diagnostic tool in the agricultural sector for disease management, considering that it can provide biological differentiation and/or similarity between fungal species.