The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depo-medroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system.