Our purpouse was to identify quantitatively and qualitatively the subgingival flora in different gestational trimesters, compared to non-pregnant women; evaluating the correlations between epidemiological characteristics, clinical diagnosis, microbiological findings and levels of estradiol and progesterone. 52 pregnant women divided into 3 groups, according to the gestational trimester and 15 non-pregnant patients, without hormonal contraceptives, were evaluated. Plaque index (PI), gingival index (GI), probing depth (PD) and clinical attachment level (CAL) were evaluated. Subgingival biofilm samples were processed by the qPCR technique and the serum levels of estradiol and progesterone quantified by chemiluminescence. Clinical diagnosis during gestation was correlated with the total bacterial count. A higher prevalence of Tannerella forsythia (Tf) was identified in first trimester of pregnancy and this periodontopathogen was correlated with the diagnosis of gingivitis among pregnant women. Porphyromonas gingivalis (Pg) showed a positive correlation with progesterone levels in the first trimester. High prevalence of periodontopathogens was noticed in this population. Clinical diagnosis in gestation was positively correlated with the total amount of bacteria, without influence of the hormonal levels or the epidemiological factors evaluated. The presence of Tf favored occurrence of gingivitis during pregnancy and the progesterone levels in the first trimester enhanced the growth of Pg.