Background: Urinary tract infection (UTI) is due to infection of microorganism involved urinary tract. Recently, antibiotics resistance rate is continually increase in UTI isolates. So, choice of antimicrobial drug becomes limited. Fosfomycin may be a promising alternative antimicrobial drug in that resistance cases in current scenario. Objective: Study focused on detects vitro sensitivity of fosfomycin against gram negative UTI isolates. Method: A study conducted at Microbiology Department of Smt. B. K. Shah Medical Institute and Research Centre, Piparia from January 2022 to October 2022. During study period total 256 patient’s urine culture found positive for microorganisms. Out of 256 bacterial isolates, 199 (77.8%) gram-negative bacteria, isolates which were included in our study. Bacterial isolates identified by standard laboratory procedure and antibiotic sensitivity was done by VITEK 2 compact system of bioMerieux. Results: Sensitivity of fosfomycin against isolated bacteria revealed that, 96%, 72.4%, 75%, 100%, 75% & 61.9% isolates were sensitive to Escherichia coli, Kebsiella pneumoniae, Proteus mirabilis, Enterobacter sp., Citrobacter sp. & Pseudomonas areuginosa respectively. Against Extended spectrum beta-lactamases (ESBLs) producing Enterobacteriaceae, fosfomycin was given good result which were 91.4%, 76.9%, 60% and 100% sensitive for Escherichia coli, Kebsiella pneumoniae, Proteus mirabilis and Enterobacter sp. respectively Conclusion: Fosfomycin showed better drug of option in most of the common Enterobacteriaceae and non fermented bacteria as well as in ESBL-producing Enterobacteriaceae |