The aim of this study was to characterize the ampelographic and genetic profiles of Vitis vinifera L. cv. Nero Antico di Pretalucente and to describe the grape-borne fungal communities. The oenological characteristics and the aroma profile of wine obtained by spontaneous fermentation were also investigated. Microsatellite profiles and ampelographic traits indicated that this cultivar presented a unique profile, and therefore it can be considered a cultivar in its own right and autochthonous of Gessopalena village. Next-generation sequencing analysis revealed that Aureobasidium spp. was the main genus detected on grapes. At the species level, Aureobasidium pullulans was the main species, followed by Alternaria alternata. Wines were characterized by a final ethanol content of 12.75% (v/v), a pH of 3.4, a volatile acidity lower than 0.6 g/L, a content of glycerol of 8.56 g/L, and a concentration of polyphenols and anthocyanins of 977 GAE/L and 266 mg/L, respectively. The intensity and tonality of the wine as well as the active odor compounds found were described. The results obtained could improve the knowledge concerning the agronomic traits and the wine obtained from this ancient and autochthonous grapevine variety cultivated in a foothill area, in order to offer consumers a wine with unique traits.