The photothermolysis of living EMT-6 breast tumor cells triggered by gold nanorods (AuNRs) with two-photon irradiation was conducted in situ and under real-time observation. The morphology and plasma membrane permeability of the cells were key indicators to the phenomena. AuNRs with an aspect ratio of 3.92, and a longitudinal absorption peak at 800 nm were synthesized with a seed-mediated method. The nanorods surfaces were further modified with polystyrenesulfonate (PSS) for biocompatibility. The prepared nanorods displayed excellent two-photon photoluminescence imaging. In situ real-time results revealed cavities internal to the cells were created from thermal explosions triggered by AuNRs localized photothermal effect. The cavitation dynamic is energy dependent and responsible for the perforation or sudden rupture of the plasma membrane. The energy threshold for cell therapy depended significantly on the number of nanorods taken up per cell. For an ingested AuNR cluster quantity N ∼ 10–30 per cell, it is found that energy fluences E larger-than 93 mJ/cm2 led to effective cell destruction in the crumbled form within a very short period. As for a lower energy level E = 18 mJ/cm2 with N ∼ 60–100, a non-instant, but progressive cell deterioration, is observed.