Our understanding of how diseases spread has greatly benefited from advances in network modeling. However, despite of its importance for disease contagion, the directionality of edges has rarely been taken into account. On the other hand, the introduction of the multilayer framework has made it possible to deal with more complex scenarios in epidemiology such as the interaction between different pathogens or multiple strains of the same disease. In this work, we study in depth the dynamics of disease spreading in directed multilayer networks. Using the generating function approach and numerical simulations of a stochastic susceptible-infected-susceptible model, we calculate the epidemic threshold of synthetic and real-world multilayer systems and show that it is mainly determined by the directionality of the links connecting different layers, regardless of the degree distribution chosen for the layers. Our findings are of utmost interest given the ubiquitous presence of directed multilayer networks and the widespread use of disease-like spreading processes in a broad range of phenomena such as diffusion processes in social and transportation systems.[11] can hinder disease spreading via peer punishment (e.g. prohibiting traveling if infected) and peer rewarding (e.g. free medical treatment from organizations). Both peer punishment and peer rewarding would deviate individual interactions far from symmetry, also inducing directionality in disease contagion. Even more, the own dynamics of the entities producing the diseases might be asymmetrical. For example, the interplay between cancer and the immune system can show asymmetric relationships [12]. There are also diseases with long latent periods that induce complicated dynamics between individuals who develop further the disease and those who do not, such as the interaction between individuals in the primary infection phase of Tuberculosis and those in the active state [13,14]. For those cases, multilayer networks might be able to help disentangling dynamics that would be otherwise hidden.The use of directed multilayer networks is not constrained to diseases that can infect human populations. Indeed, analogous scenarios can be found in the interface between wildlife and livestock, with diseases being endemic in one of them and then being transmitted unidirectionaly to the other [15]. This directionality is particularly relevant in the surveillance of diseases within the livestock industry, where the direction of the livestock interchange between farms can uncover structural changes that would be otherwise hidden [16]. Even more, the recent introduction of high resolution data of face-to-face interactions has also renewed the interest in using directed networks both in human and animal populations [17,18]. This data can be used to build temporal multilayer networks in which the connections between layers, i.e. different time frames, have to be necessarily directed in order to preserve the causality induced by time ordering [19].In this work, we aim at characterizing...