When Timing Matters - Shedding light on mechanisms under-lying host-pathogen dynamics in freshwater Invertebrates possess an innate immune system that acts non-specifically against pathogens and is regulated by the circadian clock. We used a well-established host–parasite system, Daphnia magna and its parasite Pasteuria ramosa, to investigate day–night differences in susceptibility. In an infection experiment where hosts were exposed to spores either during day or night, we found slight increases in daytime infection. We demonstrate that Daphnia exhibits rhythmic expression of five immune genes, with a major peak around the transition from day to night and low expression during day likely explaining higher susceptibility during day. Higher daytime susceptibility may benefit Pasteuria, as Daphnia likely encounters surface sediments with spores during day due to diel vertical migration. Exposure to the clock-dependent hormone melatonin altered immune gene expression and increased susceptibility both day and night, probably by immune suppression from melatonin. Melatonin may influence the parasite by signaling spore maturation. The freshwater keystone species Daphnia may experience altered infection dynamics when circadian rhythms and melatonin signaling are disrupted by anthropogenic stressors, with potential consequences for population structure and food webs. Our results spotlight the mechanisms underlying infection risk in host-pathogen systems highlighting the importance of circadian regulation for disease dynamics in freshwater ecosystems.