Microorganisms are versatile agents that modify and shape the environment in which we live. Because they have a very diverse metabolic repertoire, there is a significant gap in knowledge on the nutritional needs of keystone microorganisms in different environments. In addition, they form complex microbial communities through their interactions with the environment and with other microorganisms and may also generate products that are often of special interest for biotechnological applications in the food and pharmaceutical industries. Therefore, understanding the behavior of microorganisms in the face of external perturbations on the ecosystem in which they live and on the presence of other organisms that could antagonize them is of crucial importance to elucidate the complex webs of interaction that shape microbial communities. Therefore, this methods collection will cover methods in microbial ecology, including culturomics, in vitro models of cell-to-cell communication, in vitro biofilm dynamics modeling, in vitro models of microbial/host interactions, and bioinformatics and computational methods for microbial interaction studies, such as metagenomics, metatranscriptomics, and metaproteomics methods to understand microbial community assemblages.