Biofilms are increasingly recognized as being the predominant form for survival for most bacteria in the environment. The successful colonization of Vibrio fischeri in its squid host Euprymna tasmanica involves complex microbe–host interactions mediated by specific genes that are essential for biofilm formation and colonization. Here, structural and regulatory genes were selected to study their role in biofilm formation and host colonization. We have mutated several genes ( pilT , pilU , flgF , motY , ibpA and mifB ) by an insertional inactivation strategy. The results demonstrate that structural genes responsible for synthesis of type IV pili and flagella are crucial for biofilm formation and host infection. Moreover, regulatory genes affect colony aggregation by various mechanisms, including alteration of synthesis of transcriptional factors and regulation of extracellular polysaccharide production. These results reflect the significance of how genetic alterations influence communal behavior, which is important in understanding symbiotic relationships.