Background: Gram-negative bacilli are primarily responsible for the most common pediatric infections. Frequently, Escherichia fergusonii is identified as E. coli because of its close genetic proximity. Objectives: We aimed at the isolation and identification of multi-resistant strains of E. fergusonii, affecting children under two months of age. Methods: Strains were isolated from infectious processes and were identified phenotypically and molecularly. The microdilution method (MicroScan, autoSCAN-4) and the disk diffusion method (modified Kirby Bauer) were used to analyze antibiotic susceptibility. Results: Strains isolated were multi-resistant. Molecular identification provided the correct taxonomic assignment. Escherichia fergusonii strains were wrongly identified as E. coli with the phenotypic identification method. In addition, Pseudomonas aeruginosa and Klebsiella pneumoniae were identified. The best sensitivity results were obtained with Ceftazidime/avibactam and ceftolozane/tazobactam. Conclusions: We provided the first report of isolation and identification of multi-resistant E. fergusonii strains affecting children under two months of age in a neonatal intensive care unit.