Mycotoxins are secondary metabolites of filamentous fungi that can cause toxic effects in human and animal health. Most of the filamentous fungi that produce these mycotoxins belong to four genera, namely, Aspergillus, Penicillium, Fusarium, and Alternaria. Mycotoxigenic fungi, along with mycotoxins, create a constant and serious economic threat for agriculture in many terms, counting product losses due to crop contamination and food spoilage, as well malnutrition when considering nutritional quality degradation. Given the importance of robust and precise diagnostics of mycotoxins and the related producing fungi in the grape food chain, one of the most important agricultural sectors worldwide, the present review initially delivers a comprehensive presentation of mycotoxin reports on grape and derived products, including a wide range of commodities such as fresh grapes, raisins, wine, juices, and other processed products. Next, based on worldwide regulations’ requirements for mycotoxins, and referring to the relative literature, this work presents methodological approaches for mycotoxin determination, and stresses major methods for the detection of fungal species responsible for mycotoxin production. The principle of function and basic technical background on the available analytical and molecular biology techniques developed—including chromatography, mass spectrometry, immunochemical-based assays, biosensors, and molecular assays—is briefly given, and references for their application to grape and derived product testing are highlighted.