Background Anaesthetic management of cancer surgery may influence tumour recurrence. The modulation of gene expression by methylation of deoxyribonucleic acid (DNA) (epigenetics) is increasingly recognized as a major hallmark of cancer. Next to direct effects of local anaesthetics upon tumour cells, the ester-type local anaesthetic, procaine, has been shown to affect methylation status in several tumour cell lines, promoting the reactivation of tumour suppressor genes. We sought to determine whether the prototype amide-type local anaesthetic, lidocaine, influences the survival and epigenetic status of oestrogen receptor (ER)-positive and -negative breast cancer cell lines in vitro. Methods Breast cancer cell lines BT-20 (ER-negative) and MCF-7 (ER-positive) were incubated with lidocaine and procaine as the reference substance. We performed cell count and determined apoptosis using TUNEL stain. Further, we assessed global methylation status, and methylation of three known tumour suppressor genes (RASSF1A, MYOD1, and GSTP1) using the MethyLight assay and real-time quantitative polymerase chain reaction, respectively. Results Baseline methylation was 100-fold higher in BT-20 cells. Here, we observed a dose-dependent decrease in DNA methylation in response to lidocaine (1, 0.01, and 0.01 mM) after 72 h (P<0.001, <0.001, and 0.004, respectively). The corresponding changes were smaller in MCF-7 cells. Global methylation status was profoundly influenced, but the methylation and mRNA expression status of three tumour suppressor genes was unchanged. Conclusions Our findings suggest that demethylating tumour-suppressive effects of anaesthetic interventions may only be detectable in specific types of cancer due to differential methylation profiles. In conclusion, at clinically relevant concentrations, lidocaine demethylates DNA of breast cancer cell lines in vitro.