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Background: The COVID-19 pandemic disrupted healthcare systems and antimicrobial stewardship, potentially altering antimicrobial resistance patterns. This study characterized temporal changes in the proportions of multidrug-resistant organisms (MDROs) and identified associated factors before and during the pandemic at a tertiary care hospital in northeastern Thailand. Methods: A single-center retrospective observational study was conducted at Roi Et Hospital, including 5458 culture-confirmed MDRO cases (2017-2022), stratified into pre-pandemic (2017-2019) and pandemic (2020-2022) periods. Pathogen-specific resistance proportions were compared using odds ratios (ORs) with 95% confidence intervals (CIs). Multivariable logistic regression identified independently associated factors within each period. Results: The proportion of MDRO cases classified as hospital-acquired increased from 40.71% to 57.41% (p < 0.001). Carbapenem-resistant Acinetobacter baumannii (CRAB) increased markedly (22.87% to 76.11%; OR 10.75, 95% CI 9.43-12.26), followed by carbapenem-resistant Enterobacterales (CRE) (4.05% to 21.61%; OR 6.54, 95% CI 5.84-7.32) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) (14.32% to 27.15%; OR 2.23, 95% CI 1.87-2.65; all p < 0.001). Vancomycin-resistant Enterococcus (VRE) declined significantly (OR 0.41, 95% CI 0.25-0.68; p < 0.001). Methicillin-resistant Staphylococcus aureus (MRSA) showed a higher proportion among clinical isolates (3.28% to 6.34%; OR 2.01, 95% CI 1.42-2.83), although without a consistent annual trend. In multivariable analyses, ICU admission was independently associated with lower odds of CRE (aOR 0.52) and CRPA (aOR 0.63) and with higher odds of CRAB (aOR 2.13; all p < 0.001). Conclusions: The COVID-19 pandemic was associated with a major proportional shift toward carbapenem-resistant Gram-negative pathogens, with distinct profiles of associated factors across CRAB, CRE, and CRPA. These findings highlight the need for pathogen-specific infection prevention and antimicrobial stewardship strategies during healthcare system disruptions.