Limitations of cancer margin delineation and surgical guidance by means of autofluorescence imaging under conditions of laser ablation were investigated and preliminary results are presented. PinPointTM (Novadaq Technologies Inc., Canada) was used to capture digital images and Er:YAG laser (2.94μm, Glissando, WaveLightTM, Germany) was exploited to cause laser ablation on both normal and cancer sites of the specimen. It was shown that changes of the autofluorescence image after ablation extend beyond the actual sizes of the ablation loci. The tumor tissue after the laser ablation starts to emit fluorescent light within the green wavelength band (490–550nm) similar to normal tissue stating that the current technology of in-process tissue classification fails. However, when the autofluorescence was collected in the red range (600–750nm), then the abnormal/normal contrast was reduced, but still present even after the laser ablation. The present study highlights the importance of finding a proper technology for surgical navigation of cancer removal under conditions of high power effects in biological tissues.