Most bacterial species are now acknowledged to live in complex structures called biofilms. Biofilm communities present serious problems in infectious diseases, such as bacterial vaginosis, cystic fibrosis, dental plate and infections associated with indwelling medical devices. Planktonic free-floating bacteria express different phenotypes as compared with bacteria inside the biofilm communities. Studying these differences is of the utmost importance in order to better understand why bacteria in biofilms are normally more pathogenic than planktonic bacteria. Analysing the genetic expression of such bacterial communities provides some challenges mainly due to the presence of extracellular polymeric substances surrounding the bacteria, what is called the biofilm matrix. This book was written for undergraduate and graduate students in the field of microbial biofilms, basic molecular biology and transcriptomics, by presenting compreensive information and detailed protocols optimized for Gram-positive and Gram- negative bacterial biofilm RNA extraction. The reader will learn how to work with complex microbial samples and how to quantify the genetic expression of genes of interest.