A 15th century St Theodoros icon of outstanding quality is on display at the Zakynthos Ecclesiastical Art Museum. On the basis of certain stylistic characteristics, this icon has been attributed to the legendary Cretan painter Angelos Akotantos. In order to explore the latter attribution, the icon was subjected to examination via multispectral imaging, while microsamples were investigated through an optical microscope (OM), a scanning electron microscope coupled with an energy dispersive analyzer (SEM-EDX), μ-Raman and X-ray diffraction (XRD). The data were evaluated in the light of the findings of recent analytical studies conducted on several genuine Angelos icons. Identified materials include gypsum, gold leaf, bole, natural ultramarine, lead white, charcoal, green earth, red lake, minium, cinnabar, and red and yellow ochres. The identified materials resemble those employed by Angelos, while the identification of ultramarine is of particular significance, as this extremely expensive and rather rare pigment was very often used by the particular painter. Moreover, multispectral imaging reveals notable painting technique similarities between the icon in consideration and known Angelos icons, while cross sections of corresponding samples exhibit almost identical structures. Overall, the present work considerably strengthens the suggestion that the St Theodoros icon in consideration was painted by Angelos and also widens our knowledge regarding the late Byzantine painting.