Background <p>Patients with diabetes mellitus are susceptible to bacterial infections and may be exposed to repeated or prolonged antimicrobial therapy, which can increase the risk of multidrug-resistant organism (MDRO)-positive cultures. However, the distribution of MDROs across different specimen types and the relationship between glycemic control and MDRO isolation remain insufficiently characterized in regional clinical microbiology settings. This study investigated specimen-specific MDRO profiles and evaluated the association between glycated hemoglobin (HbA1c) levels and MDRO-positive cultures in adults with diabetes.</p> Methods <p>This retrospective clinical microbiology study was conducted at a regional teaching hospital in southern Taiwan. Culture-positive isolates obtained from adult patients between January and December 2023 were reviewed. After predefined exclusions, 1989 isolates from abscess, urine, sputum, and blood specimens were included. The analysis focused on six predefined clinically monitored MDRO phenotypes routinely reported by the hospital clinical microbiology laboratory and infection-control program. Diabetes status was determined from documented physician diagnosis in the electronic medical record and/or laboratory evidence consistent with national diagnostic criteria. Poor glycemic control was defined as HbA1c &gt; 8.0%. Bacterial identification and antimicrobial susceptibility testing were performed using the VITEK-2 Compact automated system, with confirmatory disk diffusion testing when indicated. Associations among diabetes status, specimen type, demographic factors, biochemical parameters, and MDRO-positive cultures were analyzed using chi-square tests, <i>t</i> tests, and logistic regression models.</p> Results <p>Among 1989 culture-positive isolates, 902 were obtained from patients with diabetes and 1087 from patients without diabetes. The overall MDRO-positive proportions were 19.5% in the diabetes group and 17.5% in the non-diabetes group. Urine was the most frequent specimen source in patients with diabetes. MDRO distribution differed significantly by specimen type. Methicillin-resistant <i>Staphylococcus aureus</i> predominated in abscess and blood specimens, vancomycin-resistant <i>Enterococcus</i> predominated in urine specimens, and carbapenem-resistant <i>Acinetobacter baumannii</i> and carbapenem-resistant <i>Pseudomonas aeruginosa</i> were frequent in sputum specimens. In patients with diabetes, HbA1c &gt; 8.0% was associated with increased odds of MDRO-positive cultures after adjustment for age, sex, and specimen type (adjusted odds ratio, 2.82; 95% confidence interval 1.84–4.32; <i>p</i> &lt; 0.001). Routine lipid markers and uric acid were not significantly associated with MDRO status.</p> Conclusions <p>Poor glycemic control was associated with MDRO-positive cultures in adults with diabetes. These findings support specimen-specific MDRO surveillance and suggest that glycemic-control information may help contextualize MDRO-positive culture findings in clinical microbiology interpretation and antimicrobial stewardship.</p>