Combined non-linear imaging techniques were used to deeply image human ex-vivo fresh biopsies of bladder as well as to discriminate between healthy bladder mucosa and carcinoma in situ. Morphological examination by two-photon excited fluorescence and second-harmonic generation has shown a good agreement with corresponding common routine histology performed on the same samples. Tumor cells appeared slightly different in shape and with a smaller cellular-to-nuclear dimension ratio with respect to corresponding normal cells. Further differences between the two tissue types were found in both spectral emission and fluorescence lifetime distribution by performing temporal- and spectral- resolved analysis of fluorescence. This method may represent a promising tool to be used in a multi-photon endoscope, in a confocal endoscope or in a spectroscopic probe for in-vivo optical diagnosis of bladder cancer.