Background: Urinary tract infection (UTI) is considered one of the most common infections occurring in different ages. The increasing emergence and rapid spread of multidrug-resistant (MDR) pathogens has led to reuse older antimicrobials like Fosfomycin. This study aimed to evaluate the activity of Fosfomycin on MDR pathogens beside its effect on biofilm formation. Methods: A total of 116 MDR Gram -negative isolates from ICU patients suffering from UTI has been included in this study. Standard microbiological tests were done to identify the isolates. Susceptibility to various antibiotics was detected by disk diffusion method. Phenotypic tests for determining various β-lactamases were done. Minimal inhibitory concentration (MIC) for Fosfomycin was detected by agar dilution method. Formation of biofilm by the isolates with and without adding Fosfomycin was assessed by microtiter plate method. Results: The most frequently isolated pathogen was E. coli (70/116); 60.3% followed by Klebsiella spp. (31/116); 26.7%. Fosfomycin showed a high level of inhibitory effect on most of tested isolates ; E. coli revealed low resistance rate of 4.2%, while Klebsiella spp, Pseudomonas aeruginosa and Acinetobacter baumani showed resistance rate of 16% ,36%), and 50%, respectively. A total of 72 (62.1%) isolates was ESBL producers, of which 92% isolates were Fosfomycin - sensitive , while 25(22%) isolates were MBL-positive, of which 88% were sensitive to Fosfomycin. Eighty-seven (75%) isolates were biofilm producers. Fosfomycin inhibited biofilm formation in 67(77%) isolates. Conclusion: ESBL and MBL producing Gram negative urinary pathogens showed high sensitivity level to Fosfomycin. Also, Fosfomycin had good inhibitory effect on their biofilm formation.