Zoonotic diseases represent a public health problem worldwide, since approximately 60% of human pathogens have a zoonotic origin. A variety of methodologies have been developed to diagnose zoonosis, including culture-dependent and immunological-based methods, which allow the identification of a huge range of pathogens. However, some of them are not detected easily with these approaches. Additionally, molecular tests have been developed, and they are designed to identify a single pathogen or mixtures of them. In this context, metagenomics comes as an alternative to get genome sequences of different microorganisms, which comprise a microbial community. Metagenomics have been used to characterize microbiomes and viromes, which are not cultivable under laboratory conditions. This methodology could be a powerful tool in the diagnosis of zoonotic diseases because it allows not only identification of genus and species, but also detection of some proteins in specific conditions on specific tissues, through structural and functional metagenomics, respectively.