The success of Azospirillum in the rhizosphere is attributed to its chemotactic response to plant signals and nutrients, as well as its capacity to undergo morphological changes, form cellular aggregates, and produce extracellular polymeric substances, which contribute to robust biofilm formation. The biofilm matrix confers Azospirillum cells with protection against antibacterial agents released by competing microorganisms and abiotic stress while facilitating root colonization to establish a mutually beneficial association with the host plant. This chapter compiles numerous studies describing the composition of Azospirillum biofilms, their regulatory mechanisms, and their role in interactions with other microorganisms. It also addresses the processes of flocculation and aggregation, highlighting their importance in the development of effective microbial inoculants. Furthermore, the chapter provides a detailed overview of attachment and root colonization mechanisms, as well as the role of colonization in plant biostimulation. Finally, standardized and detailed protocols for the assessment of biofilm formation, flocculation, surface attachment, and root colonization by Azospirillum are provided.