Candida albicans is an opportunistic human pathogen that can become virulent due to its ability to switch from a benign yeast to virulent hyphae phenotype. The emergence of C. albicans resistant to commonly used antifungal agents has necessitated the development of innovative treatments, which must be accompanied by an understanding of the molecular changes that occur during the phenotypic shift. For this purpose, Fourier transform mid-infrared spectroscopy in attenuated total reflectance mode (FT-mIR-ATR) was applied to monitor the structural and compositional changes in C. albicans during the yeast-to-hyphae transition. FT-mIR-ATR measurements were completed on the whole cell of C. albicans (SC5314) during hyphal formation induced by N-acetylglucosamine at 0, 1, 2, 3, 6 and 24 h. Principal component analysis separated the FT-mIR-ATR spectra into four groups that were aligned with the morphological changes captured by microscopic imaging. Spectral signatures indicating the structural and compositional modifications during the transition were identified mainly in the fatty acid region (3100-2800 cm-1), the protein and peptide region (1800-1500 cm-1), the mixed region (1500-1200 cm-1) and the polysaccharide region (1200-900 cm-1). A spectral fingerprint of the transition via a heat map was generated based on the peak shift in position. Quantitative evaluation of the spectra by curve fitting further revealed the dynamics of the cell's main components during the transition. This work provides valuable structural and functional information on the C. albicans phenotypic transition to hyphae, which has diagnostic implications.