Biofilms complete a life cycle where cells aggregate, grow and produce a structured community before dispersing to colonize new environments. Progression through this life cycle requires temporally controlled gene expression to maximize fitness at each stage. Previous studies have largely focused on identifying genes essential for the formation of a mature biofilm; here, we present an insight into the genes involved at different stages of biofilm formation. We used TraDIS-Xpress, a massively parallel transposon mutagenesis approach using transposon-located promoters to assay the impact of disruption or altered expression of all genes in the genome on biofilm formation. We identified 48 genes that affected the fitness of cells growing in a biofilm, including genes with known roles and those not previously implicated in biofilm formation. Regulation of type 1 fimbriae and motility were important at all time points, adhesion and motility were important for the early biofilm, whereas matrix production and purine biosynthesis were only important as the biofilm matured. We found strong temporal contributions to biofilm fitness for some genes, including some where expression changed between being beneficial or detrimental depending on the stage at which they are expressed, including dksA and dsbA. Novel genes implicated in biofilm formation included zapE and truA involved in cell division, maoP in chromosome organization, and yigZ and ykgJ of unknown function. This work provides new insights into the requirements for successful biofilm formation through the biofilm life cycle and demonstrates the importance of understanding expression and fitness through time.