In previous cross-sectional or case-control studies, clinical periodontal disease has been associated with gestational diabetes mellitus. To test the hypothesis that, in comparison with women who do not develop gestational diabetes mellitus, those who do develop it will have had a greater exposure to clinical and other periodontal parameters, we measured clinical, bacteriological (in plaque and cervico-vaginal samples), immunological, and inflammatory mediator parameters 7 weeks before the diagnosis of gestational diabetes mellitus in 265 predominantly Hispanic (83%) women in New York. Twenty-two cases of gestational diabetes mellitus emerged from the cohort (8.3%). When the cases were compared with healthy control individuals, higher pre-pregnancy body mass index (p = 0.004), vaginal levels of Tannerella forsythia (p = 0.01), serum C-reactive protein (p = 0.01), and prior gestational diabetes mellitus (p = 0.006) emerged as risk factors, even though the clinical periodontal disease failed to reach statistical significance (50% in those with gestational diabetes mellitus vs. 37.3% in the healthy group; p = 0.38).