The interaction between gold nanoparticles and femtosecond laser irradiation has been shown to be effective in damaging malignant cells in two-dimensional cell cultures. Further studies with rodent models have also shown promising results; however, rodent physiology and genetics are known to differ substantially from that of humans, often failing to successfully predict the human response to treatment. For bridging the gap between two-dimensional cell cultures and animal models for plasmonic phototherapy, we use a natural hydrogel extracted from mouse sarcoma as a three dimensional scaffold for growing breast cancer epithelial cells in co-culture with healthy fibroblast cells. The malignant cells were specifically targeted using anti-EGFR-coated gold nanospheres, followed by washout of unbound particles and irradiation by amplified femtosecond pulses at off-resonance wavelength of 800 nm. Irradiated cell colonies seized to develop and grow; after approximately three days, time-lapse imaging revealed widespread death of both normal fibroblasts and malignant epithelial cells, leading to disintegration of all cell colonies. Pulse broadening experiments demonstrated similar cell death for pulses between 45 fs and 400 fs of equal energy, while pulses longer than 500 fs had no visible effect on the cells. The results suggest that cell damage is primarily photothermal, and that cells in three-dimensional cultures are more resistant to the effect of the laser pulses, requiring several irradiation sequences and exhibiting slow colony disintegration.