Abstract Background Oxygen availability and extracellular acidity both have a strong impact on growth and cultivation characteristics of eukaryotes, however they are often considered in isolation, whereby a single parameter is varied at a time to identify its impact, rendering the investigation of synergistic effects created by two or more factors non-achievable. This study identified the synergistic effect between environmental pH and oxygen levels on the physiological and cellular characteristics of the simplest eukaryote, Saccharomyces cerevisiae . Materials and methods The physiological, transcriptomic, and metabolic responses of yeast were investigated during batch growth in a 2 × 2 factorial design setting; environmental pH and oxygen availability were either controlled at their optimal settings, or allowed to follow their own course during cultivation. Results Synergistic effects had a significant impact on yeast physiology, which was provoked further by both the modulation of gene expression by transcription, and the modification of metabolite pools. Genes involved in cytoplasmic translation, the extracellular and intracellular amino acid and their precursor metabolite pools were significantly responsive to concurrent variations in these two factors. Conclusion The synergistic effect of extracellular acidity and oxygenation on eukaryotic landscape of growth-associated events was significantly more pronounced than their individual effects.