Artificial light at night (ALAN) can disrupt numerous biological processes, and is increasingly studied in animal models. Here, we evaluated the impact of red and blue ALAN on Drosophila melanogaster, focusing on fertility, development, circadian rhythms, and gene expression. All results were compared to those of a control group maintained under a 12 h white light/12 h dark cycle. Red ALAN exposure increased the number of eggs laid but reduced the hatching rate and shortened the larval period. Conversely, blue ALAN led to fewer eggs laid, fewer emerging adults, and lower hatching success. Significant alterations in circadian rhythm and the sleep-wake cycle were observed in flies exposed to both red and blue ALAN, including a reduction in mean locomotor activity over 24 h and during the daytime period, increased sleep duration during the day, and reduced sleep duration at night. Effects were more pronounced under blue ALAN, which disrupted circadian rhythm by eliminating morning and evening activity peaks and increasing nocturnal activity. Gene expression analyses revealed that red ALAN upregulated ecdysone-induced protein 74EF (E74) and the ecdysone receptor (EcR) expression in adults, while juvenile hormone binding protein 1 (Jhbp1) was elevated under both light conditions. In larvae, both ALAN spectra increased expression of E74 and Jhbp1. These findings demonstrate that red and blue ALAN can significantly disrupt fertility and development in Drosophila melanogaster. Given the rising prevalence of light pollution and night-shift work, further studies are needed to investigate ALAN-related reproductive impairments in other animals, including vertebrates and humans.