Mycotoxins are toxic metabolites produced by fungus that can be parasites or saprophytes of crops or livestock forage. Consumer demand for plant-based foods and interest in animal-based foods originating from animals fed plant-based feed has been on the rise. Therefore, monitoring mycotoxins occurring in the food supply is more critical than ever. The goal of this project is to improve surface-enhanced Raman spectroscopy’s (SERS) ability to identify and detect mycotoxins using label-free SERS substrates. Two simple approaches were designed to enhance the detection of mycotoxins produced by the Aspergillus and Penicillium genera, ochratoxin A and aflatoxin B1. Ochratoxin A was successfully detected in wine samples spiked with the mycotoxin in a range of 0.01 to 1 ppm using a facile solvent-mediated extraction that showed the key role that the food matrix can play on the SERS substrate performance. The detection of aflatoxin B1’ SERS signals using bare gold nanoparticles was enhanced with the addition of human serum albumin (HSA) as a mediating molecule. A combination of the HSA-mediated protocol and a liquid-liquid extraction (LLE) method allows the detection of up to 2 ppb of AFB1 in compound feedstuff samples. Additionally, a simple SERS protocol applied to Aspergillus flavus grown in liquid and solid medium showed the technique’s capacity to classify between aflatoxigenic and non-aflatoxigenic species. Raman spectroscopy, SERS, Infrared Spectroscopy (IR) and surface-enhanced infrared absorption spectroscopy (SEIRAS) showed differences yet potential complementarity in their ability to identify mycotoxins produced by the Fusarium genus, deoxynivalenol and fumonisin B1.