Introduction: Chlamydia trachomatis (CT) is a sexually transmitted bacterium that is highly prevalent in young patients. Chlamydial infections during the gestational period have been associated with adverse obstetric outcomes, such as spontaneous preterm birth (sPTB). However, results in the literature are inconclusive. Objective: To evaluate the association between CT infection and sPTB. Methods: This was a case-control study nested in the cohort of the prospective Brazilian Ribeirão Preto and São Luís birth cohort study. Pregnant patients were recruited in private and public health clinics in São Luís and Ribeirão Preto, Brazil. At the gestational age of 20-25 weeks, cervicovaginal fluid samples were collected for the diagnosis of CT using a polymerase chain reaction. Plasma levels of Transforming Growth Factor-α (TGF- α), Interferon-γ (IFN- γ), Interleukin-10 (IL-10), IL-13, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, Tumor Necrosis Factor-α (TNF- α), and TNF-β were measured using a multiplex assay. Results: Of the 561 pregnant patients evaluated, 121 had sPTB and 440 had a full-term delivery (control group). According to our results, CT infection was not associated with sPTB (odds ratio, 1.13; 95% confidence interval, 0.50–2.56); however, it was more frequent among younger patients (p=0.0078), unmarried patients (p=0.0144), and those with multiple sexual partners (p=0.0299). There were no significant differences in the immune mediators between patients with sPTB or full-term deliveries, or between patients with or without a CT infection. Conclusion: In conclusion, CT infection was not associated with sPTB in our study. However, its correlation with younger pregnant patients suggests that these patients require careful clinical management.