Toll-like receptors (TLRs) are major players of the innate immune system. Once activated, they trigger a signalling cascade that leads to NF-κB translocation from the cytoplasm to the nucleus. Single cell analysis shows that NF-κB signalling dynamics are a critical determinant of transcriptional regulation. Moreover, the outcome of innate immune response is also affected by the cross-talk between TLRs and estrogen signalling. Here, we characterized the dynamics of TLR5 signalling, responsible for the recognition of flagellated bacteria, and those changes induced by estradiol in its signalling at the single cell level. TLR5 activation in MCF7 cells induced a single and sustained NF-κB translocation into the nucleus that resulted in high NF-κB transcription activity. The overall magnitude of NF-κB transcription activity was not influenced by the duration of the stimulus. No significant changes are observed in the dynamics of NF-κB translocation to the nucleus when MCF7 cells are incubated with estradiol. However, estradiol significantly decreased NF-κB transcriptional activity while increasing TLR5-mediated AP-1 transcription. The effect of estradiol on transcriptional activity was dependent on the estrogen receptor activated. This fine tuning seems to occur mainly in the nucleus at the transcription level rather than affecting the translocation of the NF-κB transcription factor.