Abstract Aim The aim of the present study was to analyze the occurrence of P orphyromonas gingivalis ( P . gingivalis ), T annerella forsythia ( T . forsythia ), T reponema denticola ( T . denticola ), and A ggregatibacter actinomycetemcomitans ( A . actinomycetemcomitans ) in patients with diabetes. Methods Periodontal and diabetic parameters and subgingival biofilm samples were obtained from 60 patients with diabetes and 62 patients without diabetes. By using polymerase chain reaction, the prevalence of red complex microorganisms ( P . gingivalis , T . forsythia , and T . denticola) and A . actinomycetemcomitans were determined. Descriptive and non‐parametric statistical analyses between groups were performed (Kruskal–Wallis, Mann–Whitney U ‐test, and Fisher's exact test). Results Patients with diabetes presented significantly higher periodontal attachment loss levels compared to patients without diabetes. Red complex microorganisms were detected in lower frequencies in patients with diabetes. The detection of A . actinomycetemcomitans was higher in patients with diabetes and periodontitis compared to systemically‐healthy patients without periodontitis ( P < 0.05). P . gingivalis was associated with periodontitis in non‐diabetic patients ( P < 0.05), whereas A . actinomycetemcomitans was associated with periodontitis in diabetic patients ( P < 0.05). Conclusions The findings of the present study indicate that there might be differences in the subgingival microbiota between diabetic and non‐diabetic patients. In addition, P . gingivalis and A . actinomycetemcomitans were associated with periodontitis in patients without diabetes and patients with diabetes, respectively.