Artificial light at night can have negative effects on human wellbeing and health. It can disrupt circadian rhythms, interfere with sleep, and participate in the progress of civilisation diseases. The aim of the present study was to explore if dim artificial light during the entire night (ALAN) can affect melatonin production and sleep quality in young volunteers. We performed two experiments in real-life home-based conditions. Young volunteers (n = 33) were exposed to four nights of one lux ALAN or two nights of five lux ALAN. Melatonin production, based on 6-sulphatoxymelatonin/creatinine concentrations in urine, and sleep quality, based on actimetry, were evaluated. Exposure to ALAN one lux during the entire night did not suppress aMT6s/creatinine concentrations but did aggravate sleep quality by increasing sleep fragmentation and one-minute immobility. ALAN up to five lux reduced melatonin biosynthesis significantly and interfered with sleep quality, as evidenced by an increased percentage of one-minute immobility and a tendency of increased fragmentation index. Our results show that people are more sensitive to low illuminance during the entire night, as previously expected. ALAN can interfere with melatonin production and sleep quality in young, healthy individuals, and both processes have different sensitivities to light.