Infected macrophages transition into aerobic glycolysis, a metabolic program crucial for control of bacterial infection. However, antimicrobial mechanisms supported by aerobic glycolysis are unclear. Methylglyoxal is a highly toxic aldehyde that modifies proteins and DNA and is produced as a side-product of glycolysis. Here we show that despite the toxicity of this aldehyde, infected macrophages generate high levels of methylglyoxal during aerobic glycolysis while downregulating the detoxification system. We use targeted mutations in mice to modulate methylglyoxal generation and show that reducing methylglyoxal production by the host promotes survival of Listeria monocytogenes and Mycobacterium tuberculosis , whereas increasing methylglyoxal levels improves control of bacterial infection. Furthermore, we show that bacteria that are unable to detoxify methylglyoxal are avirulent and experience up to 1000-fold greater genomic mutation frequency during infection. Taken together, these results suggest that methylglyoxal is an antimicrobial innate immune effector that defends the host against bacterial pathogens.