Antibiotic resistance and biofilm formation are becoming more common in uropathogenic Escherichia coli (UPEC). Hence, the study aims to determine the antibiogram of commonly prescribed antibiotics and assess prodigiosin’s antibacterial activity against UPEC. During the study, 175 UPEC isolates were identified biochemically, and their antibiogram was studied by the VITEK 2 system. Prodigiosin was extracted from Serratia marcescens MTCC 97. The MIC of prodigiosin against UPEC strains was detected by the microbroth dilution method. The majority of the UPEC strains ( n = 135) had MIC between 15 and 30 mg/mL. No significant association was observed between the MIC of prodigiosin and the antibiogram. Biofilm assay was performed by the microtiter plate method using media with and without added prodigiosin. In media without prodigiosin, most UPEC isolates were nonbiofilm formers (NBF—55.42%), followed by weak (21.14%), moderate (MBF—13.71%) and strong biofilm formers (SBF—9.7%). When the same test was performed in media with added prodigiosin, NBF decreased to 30.28%, while SBF, MBF and WBF increased to 20%, 26.85% and 22.85%, respectively. This change in biofilm production was statistically significant ( p < 0.05, Wilcoxon signed‐rank test). The effect of prodigiosin on fim H virulence gene was evaluated using PCR. The fim H gene was present in 159 (90.85%) isolates cultured in medium devoid of prodigiosin, whereas in prodigiosin‐containing media, 132 (83.01%) isolates were positive for the fim H gene and 27 (16.98%) were negative ( p < 0.05, McNemar’s test), suggesting that the fim H‐negative isolates had either considerable suppression of the gene’s transcription or gene expression pathways. Further, biofilm production increased when prodigiosin inhibited the fim H gene, suggesting a gene‐dependent reaction that may have compelled UPEC to adopt a stress–response phenotype that favours nonspecific surface attachment to form biofilm for survival. Therefore, the effect of prodigiosin on biofilms seems to be associated with the expression of fim H, indicating a gene‐dependent response.