In this work, we investigate the non-ablative, non-thermal photo-modification of collagen fibers by femtosecond Ti:Sa laser. The effect was induced and simultaneously registered during the repetitive laser scanning of type I collagen (rat tail and bovine Achilles' tendon), and bovine cornea. An irreversible increase in two-photon autofluorescence and a decrease in second harmonic generation intensities were associated with the collagen femtosecond laser photo-modification. Confocal spectral imaging revealed the formation of new fluorescent species. Controllable nonlinear photo-modification of collagen fibers and bovine cornea with ∼2 μm spatial resolution was demonstrated.