Background: Recent investigations have demonstrated a positive association between periodontitis and pregnancy complications. The purpose of this study was to determine the effect of periodontitis and the subgingival microbial composition on preeclampsia. Methods: A case‐control study was carried out in Cali, Colombia that included 130 preeclamptic and 243 non‐preeclamptic women between 26 to 36 weeks of pregnancy. Sociodemographic data, obstetric risk factors, periodontal status, and subgingival microbial composition were determined in both groups. Preeclampsia was defined as blood pressure ≥140/90 mm Hg, and ≥2+ proteinuria, confirmed by 0.3 g proteinuria/24 hours of urine specimens. Controls were healthy pregnant women. Odds ratios (ORs) for periodontitis and subgingival microbiota compositions were calculated. Results: A total of 83 out of 130 preeclamptic women (63.8%) and 89 out of 243 controls (36.6%) had chronic periodontitis (OR: 3.0; 95% confidence interval (CI): 1.91 to 4.87; P <0.001). Clinical attachment loss increased in the case group (4.0 ± 0.10 mm) compared to the control group (3.0 ± 0.08 mm) ( P <0.001). The average newborn birth weight from preeclamptic mothers was 2.453 g, whereas in controls was 2.981 g ( P <0.001). Two red complex microorganisms, Porphyromonas gingivalis and Tannerella forsythensis , and the green complex microorganism Eikenella corrodens were more prevalent in the preeclamptic group than in controls ( P <0.01). Conclusions: Chronic periodontal disease and the presence of P. gingivalis, T. forsythensis , and E. corrodens were significantly associated with preeclampsia in pregnant women. Further research is needed to establish pathogenic mechanisms of active periodontal disease and subgingival periodontopathogens related to preeclampsia development.