Vitamin B₆ (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5-phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (¹O₂), cercosporin. We measured the rate constants (k_q) for the quenching of ¹O₂ phosphorescence by 1–4 in D₂O. The respective total (physical and chemical quenching) k_q values are: 5.5 × 10⁷ M⁻¹ s⁻¹ for 1; 7.5 × 10⁷ M⁻¹ s⁻¹ for 2, 6.2 ×10⁷ M⁻¹ s⁻¹ for 3 and 7.5 × 10⁷ M⁻¹ s⁻¹ for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased ∼10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by ¹O₂, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize ¹O₂. This photodegradation was completely stopped by azide, a known physical quencher of ¹O₂. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B₆ vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased ∼10 times in organic solvents such as ethanol and 1,2-propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1–4 in Cercospora to understand better the interactions of pyridoxine and ¹O₂ in the living fungus.