Optical-based technologies offer significant advantages compared with conventional methods for detecting mycotoxin and fungal contamination in agricultural and food commodities, such as rapidness and non-destructiveness. Hyperspectral imaging (HSI) integrates traditional imaging and spectroscopy technologies and thus makes it possible for high-throughput screening analysis in an onsite or on-line manner. Currently, HSI, in tandem with modern chemometrics, has demonstrated interesting and promising results for the detection of mycotoxin and fungal contamination in varieties of agricultural products. Therefore, the objective of this chapter is to give an overview of current research advances of HSI in both fluorescence and reflectance modes for the evaluation of mycotoxin and fungal contamination in agricultural and food commodities. Advances of HSI in evaluation of the main mycotoxins, including aflatoxins, ochratoxins, deoxynivalenol, fumonisins and their related fungal contaminants, are reviewed, and the results obtained from different studies are compared and discussed. Perspectives on its future trends and challenges concerning mycotoxin and fungal evaluation are also addressed.