This study assessed the melatonin content of six tropical fruits and examined whether human consumption could contribute to dietary melatonin as measured by 6-sulfatoxymelatonin (aMT6-s, a marker of circulating melatonin in the body). Melatonin was extracted using methanol and analyzed by high-performance liquid chromatography. In a clinical crossover study, 30 healthy volunteers consumed selected fruits one at a time, with a 1week wash-out period between fruits, until completing all six fruits. Most fruits had moderate melatonin content. Significant increases in urine aMT6-s concentrations were seen after the consumption of pineapple (266%, p = 0.004), banana (180%, p = 0.001), and orange (47%, p = 0.007). The need to analyze melatonin both in fruit and as in vivo uptake was demonstrated. Further study is warranted regarding the clinical effect of fruit consumption in people with age-related melatonin reduction problems such as sleeplessness and illnesses involving oxidative damage.