Urinary tract infection (UTI) is defined as a disease caused by microbial invasion of the urinary tract. Recently, antibiotics resistance rate is continually increase in urinary tract 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 UTIs isolates. Method: A study conducted at Department of Microbiology, 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. Identification of isolates was done by standard laboratory methods and antimicrobial susceptibility was done by automated VITEK 2 compact system. Results: Sensitivity of fosfomycin to isolated bacteria revealed that, 96%, 72.4%, 75%, 100%, 75% & 61.9% isolates were sensitive to E. 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 E. 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.