Uropathogenic Escherichia coli (UPEC), which accounts for 85% of urinary tract infections (UTI), assembles biofilms in diverse environments, including the host. Besides forming biofilms on biotic surfaces and catheters, UPEC has evolved an intracellular pathogenic cascade that culminates in the formation of biofilm-like intracellular bacterial communities (IBCs) within bladder epithelial cells. Rapid bacterial replication during IBC formation augments a build-up in bacterial numbers and persistence within the host. Relatively little is known about factors mediating UPEC biofilm formation and how these overlap with IBC formation. To address this gap, we screened a UPEC transposon mutant library in three in vitro biofilm conditions: Luria broth (LB)-polyvinyl chloride (PVC), YESCA (yeast extract-Casamino Acids)-PVC, and YESCA-pellicle that are dependent on type 1 pili (LB) and curli (YESCA), respectively. Flagella are important in all three conditions. Mutants were identified that had biofilm defects in all three conditions but had no significant effects on the expression of type 1 pili, curli, or flagella. Thus, this approach uncovered a comprehensive inventory of novel effectors and regulators that are involved in UPEC biofilm formation under multiple conditions. A subset of these mutants was found to be dramatically attenuated and unable to form IBCs in a murine model of UTI. Collectively, this study expands our insights into UPEC multicellular behavior that may provide insights into IBC formation and virulence.