Background: Antimicrobial resistance (AMR) is a major public health concern. Urinary tract infections (UTIs) account for up to 85–90% of community-acquired cases. The COVID-19 pandemic disrupted healthcare access and may have influenced resistance patterns. In this context, we retrospectively evaluated the antibiotic resistance dynamics of various bacterial strains isolated between 2019 and 2023 in a hospital unit; Methods: A total of 8217 clinical specimens (urine, wound secretions, sputum, pharyngeal exudate, nasal exudate, tracheal secretions, vaginal and cervical secretions, puncture fluids, purulent secretions, blood, ear secretions, eye secretions) were processed using standard microbiological techniques. Pathogen identification and susceptibility testing were performed with the VITEK 2 Compact system, following CLSI guidelines. Results: Following the analysis of 8217 clinical samples collected over a five-year period (2019–2023), a total of 2900 microorganisms were isolated and identified. Among these, the most frequently encountered were E. coli strains, with 1204 isolates. Urine cultures represented 71.3% of all processed samples. Out of these 5860 urine cultures, 1530 (26%) were positive. The resistance of E. coli strains to ampicillin (48–55.2%), trimethoprim/sulfamethoxazole (22.9–34%), and ciprofloxacin (21.4–31.5%) remained high throughout the period. ESBL-producing strains peaked at 17.6% in 2020, with multidrug resistance rates ranging from 14% to 22.4%. Conclusions: E. coli strains displayed persistently high resistance to ampicillin, trimethoprim/sulfamethoxazole, and ciprofloxacin, with peaks in ESBL production and multidrug resistance during the COVID-19 pandemic. These trends underscore the importance of continuous surveillance and antibiotic stewardship, with direct implications for empirical UTI therapy and broader strategies to mitigate the public health impact of antimicrobial resistance.