Melatonin, an endogenous indolamine derived from tryptophan, is primarily synthesized by the pineal gland in mammals and regulated by a complex neural system. Its release follows a circadian rhythm, which is crucial for regulating physiological processes in response to light-dark cycles in both humans and animals. In this review, we report that the presence of this hormone in bovine milk, with significant differences in concentration between daytime and nighttime milking, has increased interest in milk as a natural source of bioactive molecules. Melatonin lowers cortisol levels at night, reduces body temperature and blood pressure, coinciding with decreased alertness and performance, acts as an antioxidant and anti-inflammatory agent, modulates the immune system, offers neuroprotective benefits, and supports gastrointestinal health by scavenging free radicals and reducing oxidative stress in dairy cows. Many factors influence the release of melatonin, such as the intensity of artificial lighting during nighttime milking, the frequency of milkings, milk yield, and genetic differences between animals. Nocturnal milking under low-intensity light boosts melatonin, potentially reducing oxidative damage and mastitis risk. Additionally, ultra-high temperature (UHT) treatment does not significantly affect the melatonin content in milk. However, further research on its stability during milk processing and storage is crucial for ensuring product efficacy. In some countries, nighttime milk with naturally elevated melatonin content is already commercialized as a natural aid for sleep. Thus, naturally melatonin-rich milk may be a promising alternative to synthetic supplements for promoting better sleep and overall well-being.