Microbial multispecies ecosystems are responsible for many biotechnological processes and are particularly important in food production. In wine fermentations, in addition to the natural microbiota, several commercially relevant yeast species may be co-inoculated to achieve specific outcomes. However, such multispecies fermentations remain largely unpredictable because of multilevel interactions between naturally present and/or co-inoculated species. Understanding the nature of such interactions has therefore become essential for successful implementation of such strategies. Here we investigate interactions between strains of Saccharomyces cerevisiae and Lachancea thermotolerans. Co-fermentations with both species sharing the same bioreactor (physical contact) were compared to co-fermentations with physical separation between the species in a membrane bioreactor ensuring free exchange of metabolites. Yeast culturability, viability and the production of core metabolites were monitored. The previously reported negative interaction between these two yeast species was confirmed. Physical contact greatly reduced the culturability and viability of L. thermotolerans and led to earlier cell death, compared to when these yeasts were co-fermenting without cell–cell contact. In turn, in the absence of cell–cell contact, L. thermotolerans metabolic activity led to an earlier decline in culturability in S. cerevisiae. Cell–cell contact did not result in significant differences in the major fermentation metabolites ethanol, acetic acid and lactic acid, but impacted on the production of some volatile compounds.