Culture media are important environmental factors in microbial growth, and strongly affect various cellular metabolic processes. This study used metabolomics to investigate the effect of minimal and complex media on the metabolic profiles of microorganisms. The intracellular metabolic profiles of Escherichia coli and Saccharomyces cerevisiae, cultivated in these media, were analyzed by gas chromatography/time-of-flight mass spectrometry. Of the 120 intracellular metabolites identified in the two microorganisms, the levels of amino acids, essential for protein synthesis and cell growth, were significantly higher in the complex than in the minimal media cultures. In contrast, the levels of sugars, sugar alcohols, and fatty acids were significantly higher in the organisms grown in the minimal than in complex media. These results suggest that the cells prepare their metabolism for survival in the minimal media whereas they are actively engaged in rapid growth in the complex media.