The aim of this study was to evaluate the in vitro activities of antimicrobial agents including fosfomycin tromethamine against Gram-negative isolates recovered from urine samples. A total of 1205 strains (781 Escherichia coli, 255 Klebsiella spp, 43 Proteus spp, 38 Pseudomonas spp, 23 Enterobacter spp, 19 Acinetobacter baumannii, 4 Citrobacter spp, 3 Morganella morganii, and 3 Serratia spp) were identified by VITEK 2 during the study period, October 2014 to October 2016. Antimicrobial susceptibilities of the strains were also evaluated using the Kirby–Bauer disk diffusion method, in accordance with the Clinical and Laboratory Standards Institute guidelines. Overall, 1080 (92.5%) of the isolates tested were susceptible to fosfomycin tromethamine. Higher resistance rates were observed among inpatients compared to outpatients. Resistance rates by strain were: 2.0% for E. coli, 4.4% for Enterobacter spp, 6.9% for Klebsiella spp, 9.4% for Proteus spp, 48.6% for A. baumannii, 56.0% for Pseudomonas spp, and 100% for Morganella morganii. All Serratia spp and Citrobacter spp strains were susceptible. The highest in vitro activity was detected for amikacin, piperacillin–tazobactam, and imipenem for all strains including ESBL-producers. Regardless of ESBL production, the excellent activity of fosfomycin against E. coli, Enterobacter spp, Serratia spp, and Citrobacter spp, indicates that the drug is a valuable therapeutic option for urinary tract infections, even those with co-trimoxazole- and ciprofloxacin-resistant isolates.