The aim of this work was to compare and explore electron microscopy and atomic force microscopy (AFM) for structure determination of cellulose whiskers and their nanocomposite with poly(lactic acid). From conventional bright-field transmission electron microscopy (TEM) it was possible to identify individual whiskers, which enabled determination of their sizes and shape. AFM overestimated the width of the whiskers due to the tip-broadening effect. Field emission scanning electron microscopy (FESEM) allowed for a quick examination giving an overview of the sample; however, the resolution was considered insufficient for detailed information. Ultramicrotomy of nanocomposite films at cryogenic temperatures enabled detailed inspection of the cellulose whiskers in the poly(lactic acid) matrix by AFM. FESEM applied on fractured surfaces allowed insight into the morphology of the nanocomposite, although rather restricted due to the metal coating and limited resolution. Detailed information was obtained from TEM; however, this technique required staining and suffered in general from limited contrast and beam sensitivity of the material.