Fluorescence cystoscopy enhances detection of early bladder cancer. Water used to inflate the bladder during the procedure rapidly contains urine, which may contain fluorochromes. This frequently degrades fluorescence images. Samples of bladder washout fluid (BWF) or urine were collected (15 subjects). We studied their fluorescence properties and assessed changes induced by pH (4 to 9) and temperature (15°C to 41°C). A typical fluorescence spectrum of BWF features a main peak (excitation/emission: , ) and a weaker (5% to 20% of main peak intensity), secondary peak (excitation/emission: , ). Interpatient fluctuations of fluorescence intensity are observed. Fluorescence intensity decreases when temperature increases (max 30%) or pH values vary (max 25%). Neither approach is compatible with clinical settings. Fluorescence lifetime measurements suggest that 4-pyridoxic acid/riboflavin is the most likely molecule responsible for urine’s main/secondary fluorescence peak. Our measurements give an insight into the spectroscopy of the detrimental background fluorescence. This should be included in the optical design of fluorescence cystoscopes. We estimate that restricting the excitation range from 370–430 nm to 395–415 nm would reduce the BWF background by a factor 2.