The industrial application of pigments of biological origin has been gaining strength over time, which is mainly explained by the increased interest of the consumer for products with few synthetic additives. So, the search for biomolecules from natural origin has challenged food scientists and technologists to identify, develop efficient and less consuming strategies for extraction and characterization of biopigments. In this task, elucidation of molecular structure has become a fundamental requirement, since it is necessary to comply with compound regulatory submissions of industrial sectors such as food, pharmaceutical agrichemicals, and other new chemical entity registrations. Molecular elucidation consists of establishing the chemical structure of a molecule, which allows us to understand the interaction between the natural additive (colorant, flavor, antioxidant, etc) and its use (interaction with the rest of the mixture of compounds). Elucidation of molecular characteristics can be achieved through several techniques, the most common being infrared spectroscopy (IR), spectroscopy or ultraviolet-visible spectrophotometry (UV-VIS), nuclear-resonance spectroscopy (MAGNETIC MRI), and mass spectrometry. This review provides the details that aid for the molecular elucidation of pigments of fungal origin, for a viable and innocuous application of these biopigments by various industries.