Metabolic engineering and synthetic biology approaches have prospered the field of biotechnology, in which the main focus has been on Escherichia coli and Saccharomyces cerevisiae as microbial workhorses. In more recent years, improving the Gram-positive bacteria Lactococcus lactis and Bacillus subtilis as production hosts has gained increasing attention. This review will demonstrate the different levels at which these bacteria can be engineered and their various application possibilities. For instance, engineered L. lactis strains show great promise for biomedical applications. Moreover, we provide an overview of recent synthetic biology tools that facilitate the use of these two microorganisms even more.