A total of 10 samples of grapes (bunches) without apparent fungal contamination were analyzed. The samples were collected during the 2017 and 2018 harvest from Suchá nad Parnou and Vrbové village in Small Carpathian region of Slovakia. Samples were sent to mycological laboratory, where they were stored at 4°C until their analysis. The objectives of this study were to gain more knowledge about mycobiota on grapes originating from Slovakia, with a focus on genus Penicillium and its ability to produce mycotoxins in in vitro conditions by thin layer chromatography method. For the isolation of fungi were used the direct plating technique on DRBC plates while surface sterilized grapes were used for endogenous mycobiota analysis. The plates were then incubated aerobically at 25 ±1 °C for one week in the dark. Overall, we isolated 818 strains belonging to 14 genera of filamentous microscopic fungi from surface mycobiota of grapes. The most frequent were genera Alternaria, Penicillium, Rhizopus, Sordaria, Aspergillus, Botrytis, Cladosporium and Epicoccum. The main occurring Penicillium species of the samples were P. expansum (60% Isolation frequency, 93% Relative density). A total of 388 isolates belonging to 12 genera were obtained from endogenous mycobiota. The most frequent and the most abundant genera were Alternaria, Cladosporium and Penicillium. From 3 different Penicillium species the most common was again P. expansum (30% IF, 92.5% RD). The selected isolates – P. citrinum, P. expansum, and P. chrysogenum were tested for their toxigenic ability. Out of 69 strains, 74% produced at least one mycotoxin as revealed by the method used here.