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Abstract The levels of the hormone melatonin fluctuate daily, with higher concentrations often found at night. These fluctuations likely influence multiple aspects of physiology, including the immune response. We demonstrated that the addition of exogenous melatonin increased the proportion of the freshwater zooplanktonDaphnia dentiferathat became infected by the fungal pathogenMetschnikowia bicuspidata, during the day but not at night. To determine the stage of this host–pathogen interaction at which melatonin may increase susceptibility, we conducted a series of laboratory experiments in which we raisedDaphniain the presence and absence of exogenous melatonin. To complete its life cycle,Metschnikowiamust encounter a foraging host, overcome the host's barrier resistance (gut wall), and evade the host's immune response (internal clearance). We quantified encounter rate by measuring the gut passage time and the number of spores that entered the gut. We also measured the number of spores that successfully entered the body cavity (barrier resistance) and the hemocyte response to spores entering the body cavity (one metric of internal clearance). Finally, we quantified the effect of exogenous melatonin on triggering molting. The addition of exogenous melatonin lengthened gut passage time and decreased the number of spores present in the gut. We found no effect of melatonin on the percentage of gut spores successfully entering the host's body cavity, nor on the hemocyte response. Melatonin is known to influence the timing of molting and hosts that molted during exposure were more likely to become infected, likely due to a decrease in barrier resistance. In a fully factorial experiment, there was a high death rate, low infection rate, and therefore no discernible effect of melatonin on molting, nor molting or melatonin on infection. Our results provide insight into the stages of infection where melatonin does and does not have significant effects.