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The present study aimed to determine the resistance mechanisms and clonal relationships among vancomycin-resistant Enterococci (VRE) isolated in AHEPA University Hospital during the pandemic year 2021. Overall, 140 clinical VRE were isolated during the study period and 44 were randomly selected for molecular analysis. A multiplex PCR was employed to detect vancomycin resistance genes (vanA, vanB, vanC, vanD, vanE, vanG) using specific primers. Additionally, pulsed-field gel electrophoresis (PFGE) was performed to assess the clonal relatedness of the selected isolates. The multiplex PCR showed that 36 of the 44 studied strains (81.9%) harbored the vanA gene whereas, 7 of 44 (15.9%) co-harbored the vanA and vanB resistance genes; one isolate had a negative PCR result. PFGE analysis unveiled 37 distinct electrophoretic patterns among the 44 VRE isolates with a similarity threshold of 75%. These patterns were clustered into 4 distinct branches. Our findings indicate a polyclonal distribution of vanA genes among the studied isolates and the notable concomitant presence of vanA/vanB genotypes. Furthermore, these results highlight the worsening that took place during the COVID-19 pandemic period regarding antibiotic resistance rates, underscoring the imperative need for stringent infection control measures and active surveillance.