| Summary: | Antimicrobial resistance is a global health threat resulting in increased morbidity and mortality. This retrospective study aimed to estimate antimicrobial susceptibility and multidrug resistance prevalence of clinical isolates in a regional hospital in Northern Greece during the last 6 years by analyzing the annual reports of the Laboratory of Microbiology. A total of 12,274 strains of certain bacteria were isolated from both hospitalized and ambulatory patients from biological products, mainly urine (range 63–78% during the study period). <i>E. coli</i> was the most frequent pathogen found (37.4%). A significant increase in the number of the main pathogens causing hospital-acquired infections (<i>Klebsiella pneumoniae</i>, <i>Acinetobacter baumannii</i>, <i>Pseudomonas aeruginosa</i>, <i>Enterococcus faecium</i>) was found in the time period of 2021–2023 compared to 2018–2020 (<i>p</i> < 0.0001). In total, 1767 multidrug-resistant bacterial strains were isolated, most of them belonging to <i>Acinetobacter baumannii</i> (36.4%) and <i>Klebsiella pneumoniae</i> (39.6%), and were located in the intensive care unit (ICU) (59.8%). Extensively drug resistance (XDR) and pan drug resistance (PDR) were significantly higher in 2021–2023 than in 2018–2020 (XDR: 641/1087 in 2021–2023 vs. 374/680 in 2018–2020 and PDR: 134/1087 in 2021–2023 vs. 25/680 in 2018–2020, <i>p</i> < 0.0001), resulting in an urgent need to establish certain strategies in order to eliminate this threatening condition.
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