Candida species are the most important fungal pathogens in humans and cause a variety of superficial and systemic diseases. Biofilm formation is a major virulence attribute contributing to Candida pathogenicity. Although the concentration and distribution of nutrients as well as antifungals across the biofilm thickness play a pivotal role in the development and persistence of Candida biofilms, only limited information is available on the latter aspects of Candida biofilms. Therefore, we attempted to characterize the diffusion coefficient (De) of common dietary sugars such as glucose, galactose, and sucrose in Candida albicans biofilms using horizontal attenuated total reflection-Fourier transform infrared spectroscopy (HATR-FTIR). Artificial Candida biofilms were formed using agarose polymers. De of three sugars tested, glucose, galactose, and sucrose in this artificial Candida biofilm model was found to be 4.08E-06 ± 3.63E-08, 4.08E-06 ± 3.70E-08, and 5.38E-06 ± 4.52E-08 cm2 s-1, respectively. We demonstrate here the utility of HATR-FTIR for the determination of diffusion of solutes such as dietary sugars across Candida biofilms.