Uropathogenic Escherichia coli (UPEC) is responsible for majority of urinary tract infections (UTIs), a disease increasingly complicated by antibiotic resistance and the ability of UPEC to establish persistent biofilms. A key driver of early aggregation in the biogenesis of biofilm formation is the self-associating autotransporter Antigen 43 (Ag43), whose passenger domain (Ag43α) mediates strong cell-cell ‘Velcro-like’ interactions that promote biofilm maturation. Ag43 is highly conserved across E. coli phylogenetic classes and its expression enhances UPEC persistence in vivo, making it an attractive target for anti-adhesin therapies. Here, we define the molecular interaction between a small protein fragment (SPF) and Ag43α and assess its efficacy at disrupting Ag43-mediated biofilm formation across clinically-relevant UPEC backgrounds. Biophysical analyses confirmed nanomolar binding affinities and revealed that SPF-7 likely targets a conserved subdomain required for Ag43 self-association. Circular dichroism and tryptophan fluorescence demonstrated that Ag43α maintains its secondary and tertiary structure in urine-like conditions, indicating that the molecular interface recognised by SPF-7 is preserved. Bio-layer interferometry further showed that SPF-7 retains its binding properties in these conditions, with only modest reductions in its affinity. Functionally, SPF-7 significantly reduced biofilm formation in multiple wild-type UPEC strains in both standard media and urine-like environments, indicating activity against native Ag43 expressed under physiological conditions. Together, these findings validate Ag43 as a structurally stable and functionally conserved anti-biofilm target and establish SPF-7 as a promising inhibitor capable of disrupting UPEC biofilm formation in environments reflective of the urinary tract.