Biofilms constitute a protected mode of growth that allows the colonizing microbial cells to survive in hostile environments, even when an antimicrobial agent is present. The scientific community has come to understand many things about the growth dynamics and behavior of microbial biofilms. It is now accepted that biofilm formation is a multifactorial process that starts with the adhesion of individual cells and (auto-)coaggregates of cells to a surface. Then, attached cells grow, reproduce and secrete insoluble extracellular polymeric substances. As the biofilm matures, biofilm detachment and growth processes come into balance, such that the total amount of biomass on the surface remains approximately constant in time. The detached cells retain the phenotype of the biofilm cells, which facilitates the colonization of neighboring surfaces. The most common practice to eliminate unwanted biofilms is the application of antimicrobial agents. However, conventional antimicrobial agents often show inefficacy in the control of biofilms. Much remains to be understood in the biofilm formation process and in the development of effective strategies for biofilm prevention and control. The articles contained in this Special Issue deal with biofilms of some important bacteria (including pathogens such asEscherichia coli,Pseudomonas aeruginosaandStaphylococcus aureus) and fungi (Candida tropicalis), providing novel insights into their formation mechanisms and implications, together with novel methods (e.g., use of chemical conjugates and combinations of molecules) that can be used to disrupt the biofilm structure and kill the colonizing cells.