Uropathogenic E. coli (UPEC) is the major cause of over 90% of urinary tract infections (UTIs). UTIs are the major common bacterial contaminations in health facilities and society settings. These infections require immediate medical attention as they cause discomfort to the diseased people. The cure of this disorder is complicated with the arrival of extended-spectrum β-lactamase (ESBL)-manufacturing pathogen. Therefore, the objective of this study is to determine the impact of two ancient antibiotics: Fosfomycin and Mecillinam. The experiment was conducted on diverse multidrug resilient clinical isolates. The challenges associated with the contaminations are compounded by the lack of appropriate chemotherapeutic agents. Several trials were conducted to investigate the vulnerability of clinical isolates like AmpC, + ESBL, TEME, and OXA 48. E.Coli is grown under intracellular and extracellular conditions of two longstanding antibiotics, namely Mecillinam and Fosfomycin. The antibiotics are viewed as disruptive against a range of Gram-negative bacteria. Our outcome demonstrates that these antibiotics show a lethal effect on some bacteria under study. Lastly, this investigation endorses the probable usage of both Mecillinam and Fosfomycin in the cure of these multidrug resistant microorganisms.