•NSB in Tuscany ranged between 17.2 (83 times brighter than the natural background) and 21.5 mpsas during moonless night.•Seasonal NSB during moonless night presented a different pattern in wet and dry season at urban sites.•NSB was affected by monthly cycle of moonlight and its effect interacted with weather conditions.•Average night sky during precipitation events was approximately six times (2 mpsas) than during dry days.•The presented analysis might be applied in ecological studies regarding light pollution effects on the environment. Light pollution generated by the excessive use of artificial light at night is an environmental and ecological concern. Artificial light at night is diffused far from the sources, for long distances by scattering in the atmosphere (skyglow), thus affecting the night sky and the biodiversity of rural and natural areas. The characterization of the level and variability of light pollution has become an important issue for several disciplines. This study analyzes light pollution in urban, rural and remote sites in Tuscany (Italy) by monitoring night sky brightness. Night sky brightness (NSB) data collected from 2016 to 2019 were analyzed to assess annual and seasonal variability at each site and between sites. The relationship between night sky brightness and moonlight and weather conditions were also analyzed. Trend analysis was also performed to evaluate the degradation of the quality of the measurement with time due to dirty accumulation on the sensor shield. NSB in Tuscany during moonless nights ranged between 17.3 and 21.8 mpsas. The monthly cycle of moonlight is the main driver of night sky brightness variability in the remote site (21.8 mpsas on moonless nights and 18.5 mpsas on full moon nights) with a slight, but significant darkening effect during rainy conditions (22.3 mpsas). In urban sites, moonlight cycle is almost completely masked by weather conditions, as it can be seen from the analysis of seasonal variability, lunar monthly cycle analysis and weather condition analysis. Average night sky brightness on rainy days might reach 15.4 mpsas (approximately 436 times brighter than the natural background), an intensity that is even higher than average night sky brightness in full moonlight at the same site (16.6 mpsas). Light pollution can reach levels that might affect nocturnal species, and therefore the study of long term variability of light pollution is important for ecological studies. Display omitted