Background: The aim of this study was to present the first domestic animal trial of nanosecond electroporation with chemotherapy and CO2 laser surgery. Methods: sixteen-year-old domestic cat with diagnosed melanoma on oral cavity was the case used in the study. Firstly, CO2 laser surgery was used for the removal of most of tumor mass. Then nanoelectrochemotherapy with bleomycin was applied including appropriate margin of healthy tissue. A 15 ± 4 kV/cm × 15 ns pulsing protocol was employed with a total of 800 ± 100 pulses. Only one session of nanoelectrochemotherapy (nanoECT) was performed. Results and conclusions: during the next two weeks areas of focal necrosis were replaced by the granulation tissue. Complete wound healing was observed four weeks after initial treatment. After 15 weeks post treatment, no local recurrence was apparent.