Fluorescence techniques have been widely used by scientists to reveal valuable information from biological samples, but in food science, small progress is known due to the complexity of the samples. In this study, two different biological samples, garlic skin (GS) and agave fibers (AF), were used to evaluate the techniques of confocal laser scanning microscopy (CLSM) and total internal reflection fluorescence (TIRF) microscopy, to obtain valuable information on the fiber size of the samples. A compositional characterization with calcofluor white in CLSM was achieved, but a superficial characterization of the samples with TIRF was made, evidencing fiber sizes of 398.67 ± 48.47 nm and 677.38 ± 76.88 nm for GS and AF, respectively. This work reveals that only an untreated sample can be used with the two techniques in the same microscope. In addition, it is possible to characterize the sample only using a spatial field of research and which valuable information about the structure of the material is found. This work provides the opportunity to use advanced fluorescence techniques for elucidation of structures shortly before studied with these techniques.