Interbacterial antagonism can significantly impact microbiome assembly and stability and can potentially be exploited to modulate microbes and microbial communities in diverse environments, ranging from natural habitats to industrial bioreactors. Here we highlight key mechanisms of interspecies antagonism that rely on direct cell-to-cell contact or diffusion of secreted biomolecules, and discuss recent advances to provide altered function and specificities for microbiome engineering. We further outline the use of ecological design principles based on antagonistic interactions for bottom-up assembly of synthetic microbial communities. Manipulating microbial communities through these negative interactions will be critical for understanding complex microbiome processes and properties and developing new applications of microbiome engineering.