Biofilm formation by bacteria is highly recognized for virulence factors resulting in their resistance to antimicrobials that lead to biofilm-associated infections. In this study, we isolated Escherichia fergusonii from soil, characterized its biofilm-associated growth, and evaluated the inhibitory potential of anti-biofilm compunds. Test isolate ACE12 was precisely identified as E. fergusonii based on the morphological and 16S rRNA gene sequence analysis. The antibiotic susceptibility pattern of ACE12 showed its resistance to β-Lactams, Aminoglycosides, Macrolides, Tetracycline, Trimethoprim, Vancomycin, & Nitrofurans and on the basis of its resistant pattern the isolate was categorized as extensively drug-resistant (XDR) bacteria. In addition, the research isolate ACE12 was found to harbor four distinct antibiotic resistant genes including dfrA1, blaTem-1, tetC, and sul1, encoding the resistant determinants for trimethoprim, β-lactam, tetracycline, and sulfonamide antibiotics, respectively. Initial screening of biofilm formation by Congo-Red Agar (CRA) and Tube method demonstrated that E. fergusonii ACE12 is a biofilm-forming bacterium. The respective biofilm was characterized by estimating the optical density (OD