Fungal flora on ripe grape and fungi characterization are very critical for assessing the risk of OTA presence in wine, a mycotoxin classified as possible carcinogen to humans. In the present work, the PCR-RFLP technique has been applied to the ITS1- 5.SS-ITS2 region of the rDNA to carry out the characterization of potential OTA producing species of Aspergillus section Nigri from different grape varieties grown in Spain. The toxin production levels of the different isolates have also been analysed and the ability of natamycin, a fungicide, to control fungal development and OTA production in vitro has been studied, as well as the interaction with environmental factors {water activity, temperature). The results obtained from in vitro cultures of 205 isolates of species in Aspergillus section Nigri showed that 74.2 o of A. carbonarius isolates and 14.3 o of A. tubingensis isolates were capable of producing OTA at different levels. No isolate of A. niger showed OTA producing capacity under the experimental conditions. A. carbonarius was the most frequently isolated ochratoxigenic species. The effect of natamycin against isolates of A. carbonarius has been studied according to different level, water activity and temperature. It can be seen that the efficacy of very low concentrations of this fungicide on fungal growth depends on water activity and temperature.