During the last couple of decades, the intricate processes involved in microbial biofilm formation have been effectively elucidated through the application of comparative genomics as a potent investigative technique, a widespread phenomenon with significant implications for human health, industry and the environment. Biofilm formation involves many genes and pathways, which can be identified through comparative genomic studies of biofilm-forming bacteria. This approach has proven invaluable for understanding the genomic and molecular foundation of biofilm generation by comparing the genomes of biofilm-producing bacteria. This chapter explored the role of genomic diversity in biofilm resilience and aimed to identify potential therapeutic targets through comparative genomic analysis, focusing on cases relevant to both pathogenic and environmental bacteria. This investigation centres on two key questions: (i) What genetic factors contribute to biofilm formation in microbial communities? (ii) How do comparative genomics enhance our understanding of the phenotypic traits of biofilm-forming organisms? Additionally, this chapter discusses the application of advanced techniques, such as single-cell genomics and metagenomics, to provide a more comprehensive understanding of biofilm behaviour. These cutting-edge techniques allow researchers to delve deeper into the genetic and molecular mechanisms underlying biofilm formation, thereby offering new insights into potential strategies for controlling or manipulating biofilms in various contexts.