Mitochondria are derived from endosymbiotic bacteria, with remnants of their proteobacterial past still evident today. These organelles replicate within cells through the process of mitochondrial fission. We reasoned that fission may mimic intracellular bacterial replication, thus serving to alert immune cells of a bacterial threat. Here we show that Toll-like receptor (TLR) signalling triggers activating post-translational modifications of the fission-promoting GTPase, Drp1, as well as Drp1-dependent mitochondrial fission in murine macrophages. Genetic and pharmacological approaches revealed that TLR-inducible fission promotes bacterial killing by macrophages, with both the mitochondrial unfolded protein response and lipid droplets playing a role in antimicrobial defence. The lysine deacetylase histone deacetylase 6 (HDAC6) constrains TLR-inducible fission in macrophages, via its actions on the mitochondrial fusion-promoting GTPase, Mfn1. Consequently, genetic targeting or inhibition of HDAC6 amplifies fission and enhances clearance of the intramacrophage pathogen Salmonella enterica serovar Typhimurium in vitro and in vivo. Fission-mediated bacterial killing is also apparent in the nematode Caenorhabditis elegans (C. elegans), highlighting the broad conservation of this host defence pathway across the animal kingdom. Agents that amplify fission may have applications as host-directed therapies to combat antibiotic-resistant bacterial infections.