Objectives Escherichia coli is one of the pathogenic bacteria that caused a nosocomial infection. Levofloxacin is one of the fluoroquinolones group antibiotics which is a broad-spectrum antibiotic that works effectively against E. coli. The mutation can happen in the bacteria which caused a resistant effect in the use of antibacterial. This study aimed at identifying mutation in gene gyrA among E. coli that were resistant to levofloxacin. Methods The susceptibility of E . coli was determined by disk diffusion. PCR and sequencing were performed to identify the mutation in gyrA. Results A total of 10 isolates showed result resistance to levofloxacin and gyrA gene mutation in the amino acid changes. Nucleotide sequence analysis revealed a point mutation in QRDR (quinolone resistance determining region) of gyrA Ser83→Leu, Asp87→Asn. The silent mutation was also found at codon Val85, Arg91, Ser111, Thr123. Conclusions Mutation in the gyrA gene affect the occurrence of bacterial resistance of E. coli to levofloxacin.