Biofilm formation constitutes an alternative lifestyle in which microorganisms adopt a multicellular behavior that facilitates and/or prolongs survival in diverse environmental niches. Biofilms form on biotic and abiotic surfaces both in the environment and in the healthcare setting. In hospital wards, the formation of biofilms on vents and medical equipment enables pathogens to persist as reservoirs that can readily spread to patients. Inside the host, biofilms allow pathogens to subvert innate immune defenses and are thus associated with long-term persistence. Here we provide a general review of the steps leading to biofilm formation on surfaces and within eukaryotic cells, highlighting several medically important pathogens, and discuss recent advances on novel strategies aimed at biofilm prevention and/or dissolution.