An important site of cellular damage in bacterial sepsis is mitochondrial DNA (mtDNA), which we proposed is caused by reactive oxygen and nitrogen species generated by activation of signaling through specific toll‐like receptors (TLR). In wild‐type (Wt) mice injected with heat‐inactivated E. coli, hepatic TLR4 and TLR2 proteins were up‐regulated with TLR‐dependent increases in transcript levels for tumor necrosis factor (TNF‐α), interleukin 6, nitric oxide synthase‐II (iNOS), and NADPH oxidase 2 (Nox2). The accompanying stress significantly depleted hepatic mtDNA despite eight‐ and fourfold increases in manganese superoxide dismutase (MnSOD) and mitochondrial transcription factor A (Tfam) expression, respectively. The identical E. coli dose generated significantly less TNF‐α, NO, and Nox2 in TLR4^−/− and TLR2/4^−/− but not in TLR2^−/− mice. TLR4^−/− and TLR2/4^−/− compared with Wt mice were protected from mtDNA oxidation but showed no Tfam up‐regulation and little copy number restoration. A critical role in the mtDNA damage was determined for TLR4‐mediated iNOS transcription through the MyD88 pathway. In Wt mice, mtDNA depletion was avoided by selective iNOS blockade, and residual mtDNA loss was linked to NF‐κB‐dependent TNF‐α expression. These data disclose the dual role of TLR4 in mtDNA damage and compensatory mitochondrial biogenic responses after innate immune activation.