Large-scale microbial-biotechnology processes for production of chemicals almost exclusively rely on pure cultures of microbial strains. Especially for extensively engineered pure cultures, process performance can be negatively affected, which can be caused by issues such as pathway imbalance, deterioration of productivity caused by genetic instability and enzyme promiscuity. An increasing number of studies demonstrate that, under ‘academic’ laboratory conditions, the use of defined co-cultures (i.e. deliberate mixtures of known microbial strains) offers unique possibilities for mitigating such drawbacks. These advantages differ for dissimilatory products, whose synthesis from one or more carbon substrates provides cells with free energy, and assimilatory products, whose synthesis requires a net input of free energy. Based on advances in experimental and theoretical research, this paper highlights how defined co-cultures can address several limitations of mono-cultures for production of low-molecular-weight compounds. From this largely academic perspective, we outline the key challenges for scaling these systems to industry, which underscore the need for innovative solutions and continued research in this area.