A total of 13 samples of grapes (bunches) without apparent fungal contamination were analyzed. The samples were collected during the 2019 harvest from Vrbové village in the Small Carpathian region of Slovakia. For the isolation of fungi were used the direct plating technique on DRBC plates. The plates were incubated aerobically at 25 ±1 °C for one week in the dark. The data obtained from the cultivation of the grape berry samples revealed a high diversity of fungal species (a total of 1044 isolates were obtained). Alternaria and Rhizopus were the main components of the wine grape mycobiota of the Vrbovský subregion at harvest time (92%, each), followed by Cladosporium (85%), Penicillium (77%), Botrytis and Epicoccum (54%, each). The most abundant genera found by descending order were Penicillium (25%), Alternaria (24%), Cladosporium (20%), and Rhizopus (12%) and only in minor percentage by Aspergillus (3%) among others. The main fungal species isolated from genera Penicillium and Aspergillus were Penicillium expansum (57% RD) and A. section Nigri (97% RD). Of 17 analyzed Penicillium strains, 65% were able to produce at least one of the six mycotoxins analyzed in in vitro conditions by means of thin-layer chromatography method: citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, and roquefortin C.