Microorganisms are widely distributed all over the Earth, inhabiting very diverse natural ecosystems, from the human body to inanimate indoor environments. Until recently, the methods most commonly used to study microbes have been culture-dependent approaches relying on the phenotypic evaluation of isolates that can grow in laboratory conditions. Given the advances in molecular biology and high-throughput DNA sequencing methodologies, scientists could expand their microbiome knowledge to microorganisms that do not grow well in the laboratory or have been considered too difficult and laborious to be cultivated. Culture-independent methods such as direct DNA sequencing can be performed for many samples at once, revealing the entire microbial profile of the samples and making possible the rapid characterization of the whole environmental microbiome. Investigating the microbiome profile of indoor environments such as hospitals, houses, offices and other buildings is of major concern because it could include a number of opportunistic, pathogenic or nosocomial microbes. Additionally, these environments could serve as reservoirs of virulence or antimicrobial resistance, which could be spread by humans or other vectors. High-throughput DNA sequencing has enabled large-scale microbiome screening for multiple indoor areas in a single analysis. Using this approach, we can easily track microorganisms in the environment and monitor microbiome composition related to hygiene processes or environment quality. Gaining such information and resolution regarding indoor microbiome analysis can lend very important assistance for epidemiological surveillance. Impact statement Research concerning the microbiome of indoor environments like hospitals, houses or buildings could have several implications for human health. Today, there is an ongoing shift in the paradigm of microbial analysis, from single isolated bacterial samples to entire microbiome profiles using high-throughput DNA sequencing methods. The use of sequencing methods in several studies has revealed an unprecedented microbial diversity in indoor environments, leading to a larger comprehension of the entire microbiome context. Here, we present a review of these microbiome studies using high-throughput DNA sequencing, including some new approaches and ideas that can be broadly applied in microbial tracking and epidemiological surveillance of indoor environments.