Background/Objectives: The iron-binding affinity of vaginal lactoferrin (Lf) reduces iron available to genital pathogens. We describe host reproductive, nutritional, infection and iron biomarker profiles affecting vaginal Lf concentration in young nulliparous and primigravid women in Burkina Faso. Subjects/Methods: Vaginal eluates from women who had participated in a randomized, controlled periconceptional iron supplementation trial were used to measure Lf using a competitive double-sandwich ELISA. For this analysis samples from both trial arms were combined and pregnant and non-pregnant cohorts compared. Following randomization Lf was measured after 18 months (end assessment) for women remaining non-pregnant, and at two antenatal visits for those becoming pregnant. Associations between log Lf levels and demographic, anthropometric, infection and iron biomarker variables were assessed using linear mixed models. Results: Lf samples were available for 712 non-pregnant women at end assessment and for 303 women seen at an antenatal visit. Lf concentrations of pregnant women were comparable to those of non-pregnant, sexually active women. Lf concentration increased with mid-upper-arm circumference, (P = 0.047), body mass index (P = 0.018), Trichomonas vaginalis (P < 0.001) infection, bacterial vaginosis (P < 0.001), serum C-reactive protein (P = 0.048) and microbiota community state types III/IV. Adjusted Lf concentration was positively associated with serum hepcidin (P = 0.047), serum ferritin (P = 0.018) and total body iron stores (P = 0.042). There was evidence that some women maintained persistently high or low Lf concentrations from before, and through, pregnancy. Conclusion: Lf concentrations increased with genital infection, higher BMI, MUAC, body iron stores and hepcidin, suggesting nutritional and iron status influence homeostatic mechanisms controlling vaginal Lf responses.