Antimicrobial resistance (AMR) is one of the most important problems threatening human health worldwide. The impact of the Coronavirus disease-2019 (COVID-19) pandemic on AMR continues to be discussed. Some researchers argue that the pandemic will increase AMR rates, while others suggest the opposite. The aim of this study was to investigate the change in AMR of Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus strains in three cross-sectional periods in Türkiye, the first one before the COVID-19 pandemic, the second and the third one during the pandemic. The change in antibiotic susceptibility in Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus strains isolated from urine, blood, and lower respiratory tract samples of patients hospitalized in intensive care units and wards of hospitals before (November 2019) and during the COVID-19 pandemic (November 2020 and July 2021) was investigated in this study. A total of 17 voluntary hospitals, members of the Antibiotic Susceptibility Surveillance Study Group (ADSI) of the Society for Clinical Microbiology Specialists (KLIMUD), participated in the study. Identification of bacteria was performed with automated bacterial identification systems (VITEK2, bioMérieux, France or Phoenix, BD, USA). Antibiotic susceptibility tests were performed in one center with the Kirby-Bauer disc diffusion method and in other centers with automated antibiotic susceptibility test systems (VITEK2, bioMérieux, France or Phoenix, BD, USA), and the results were evaluated according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Antibiotic susceptibility ratios were statistically analyzed using either the chi-square or Fisher’s exact test. A p-value of < 0.05 was considered statistically significant. Antibiotic susceptibility test results of a total of 4030 strains; 1152, 1139, and 1739 belonging to November 2019, November 2020, and July 2021, respectively; were examined. While cefotaxime and ceftazidime susceptibility rates in E.coli strains increased during the pandemic period compared to previous period (p= 0.04, p= 0.001, respectively); nitrofurantoin sensitivity (p= 0.02) and extended-spectrum beta-lactamase (ESBL) ratios (p< 0.001) were found to be decreased. It was determined that the susceptibility rates of all other examined antimicrobials did not change statistically. It was observed that the susceptibility rates of all antibiotics in K.pneumoniae isolates decreased during the pandemic period, but the ESBL rates increased between 2019-2020 (p= 0.01) and decreased between 2020-2021 (p= 0.02). It was found that ESBL rates increased before and after the pandemic. It was observed that the susceptibility to ciprofloxacin (p= 0.0001), levofloxacin (p= 0.003), and gentamicin (p= 0.005) in S.aureus strains increased during the pandemic period. No significant changes were observed in other antibiotic susceptibility rates. Methicillin resistance of S.aureus (MRSA) strains decreased between 2019-2020 (p= 0.03) and increased again in 2021 (p= 0.04) and returned to the pre-pandemic rate. Our study results suggest that the measures taken to reduce the spread of COVID-19 with the pandemic (such as quarantine practices, increased hand hygiene, mask use, and national/international travel restriction) may reduce the spread of bacteria such as ESBL-producing E.coli and the rate of MRSA, which is considered as a hand hygiene indicator. The increase in the later stages of the pandemic recalls the relaxation in compliance with hand hygiene rules. The decrease in the susceptibility rate of K.pneumoniae isolates to antibiotics and the increase in the ESBL rate may be due to inappropriate and excessive use of antibiotics during the pandemic period. However, we believe that these data should be supported by studies to be conducted nowadays when all the rules and measures are back as if the pandemic has ended.