A simple, rapid, sensitive and robust gas chromatographic method was developed for the simultaneous determination of free volatile carboxylic acids (FVCA) in cheese and bacterial cultures. The target analytes were extracted and converted directly from the aqueous phase to their ethyl esters using headspace. The lower detection limits for the volatile carboxylic acids in the cheese samples were less than 0.3 and less than 0.6 µmol kg−1 in the bacterial culture samples. The lower limits of quantitation in cheese were better than 0.001 mmol kg−1 for all analytes. The upper limits of quantitation varied from 39 to 136 mmol kg−1 in cheese and 78 to 272 mmol kg−1 in bacterial cultures depending on the analyte. The Horwitz ratio showed good precision for all analytes (less than 0.77). The proposed method is suitable for the determination of target metabolites directly from aqueous extracts and can also be validated for other matrices.