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The development of experimental conditions for rapid bacterial discrimination using fluorescence spectroscopy fingerprinting is presented. Colonies of Pseudomonasand related reference strains on agar plates were analyzed directly using an optic fiber coupled to a laboratory spectrofluorimeter. Spectra were collected using either classic fluorescence spectroscopy after excitation at 250 nm and 340 nm for aromatic amino acids and nucleic acids (AAA + NA) and nicotinamide adenine dinucleotide (NADH) respectively, or synchronous scanning in the excitation wavelength range 250–500 nm. Factorial discriminant analysis (FDA) showed 100% correct classification at the genus and species level from NADH spectra and 100% correct classification at the genus and species level for ∆λ= 30, 70, 90 and 110 nm (cross-validation). Analysis of variance (ANOVA) confirmed that culture time (48 or 72 h) colony and optic fiber positioning had non-significant impacts on differences between species. The use of optical fiber-fluorescence spectroscopy for bacterial discrimination directly on colonies is fast, simple and reliable. The results are independent of culture growth phase and neither need reagent addition nor prior manual preparation of cells, thus eliminating all risk of human error or contamination during sample work-up.