Mitochondria and lipid droplets (LDs) have important roles in innate immunity. Mitochondria are dynamic organelles, changing their state from a fused network (fusion) to undergoing fragmentation (fission), with this affecting cellular metabolism. Mitochondria and LDs interact during cell metabolism, demonstrating that these organelles have an intimate relationship. This study aimed to investigate the relationship between mitochondrial fission and LDs in macrophages, since both organelles contribute to antibacterial responses in these cells. I demonstrated that the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) induced mitochondrial fission and LD synthesis in both murine and human macrophages. To determine whether there is a functional link between fission and LD formation in bone marrow-derived macrophages (BMM), LPS-inducible mitochondrial fission was genetically or pharmacologically inhibited. Both regimes abrogated LPS-induced LD formation, suggesting that LPS-inducible fission drives LD formation. Signalling mechanisms driving LPS-inducible LDs are poorly understood. One potential candidate is the lysine deacetylase HDAC7 which is required for TLR-inducible fission. LPS-inducible LD formation was abrogated by either genetic or pharmacological targeting of HDAC7, whereas overexpression of HDAC7 in primary macrophages was sufficient to induce mitochondrial fission and LD formation. In bacterial infection studies, challenge with Escherichia coli (E. coli) increased both mitochondrial fission and LD formation. Interestingly, infection with Salmonella enterica serovar Typhimurium (S. Typhimurium) didn’t induce mitochondrial fission but induced LD formation, indicating a potential form of pathogen manipulation. Finally, whereas LPS-inducible fission and LD formation required HDAC7, these responses occurred independently of HDAC7 in macrophages responding to bacterial challenge. These data further support a connection between fission and LD formation and suggest that distinct mechanisms can initiate inducible fission and LD formation, depending on the nature of the stimulus. Collectively, my data suggest that the antimicrobial effects of mitochondrial fission in macrophages may be mediated by inducible production of antibacterial LDs.