Data on immune mediators in the genital tract and the factors that modulate them in Sub-Saharan women are limited. Cervicovaginal lavage (CVL) samples from 430 sexually active women from Kenya, South Africa and Rwanda were analyzed for twelve soluble immune mediators using Bio-Plex™ and Meso Scale Discovery multiplex platforms as well as single ELISAs. Ten bacterial species were quantified in vaginal swab samples. Bacterial vaginosis (BV) was defined by Nugent scoring. CVL from HIV-infected women showed a clear-cut pro-inflammatory profile. Pregnant women, adolescents and women engaging in traditional vaginal practices differed in specific soluble markers compared to reference groups of adult HIV-negative women. Cervical mucus, cervical ectopy, abnormal vaginal discharge and having multiple sex partners were each associated with an increase in inflammatory mediators. Interleukin (IL)-1α, IL-1β, IL-6, IL-12(p70) and IL-8 were elevated while the IL-1RA/(IL-1(α+β) ratio decreased in women with BV. Interferon gamma-induced protein (IP)-10 was lower in BV-positive compared to BV-negative women suggesting its suppression as a potential immune evasion mechanism by BV-associated bacteria. Lactobacillus crispatus and Lactobacillus vaginalis were associated with decreased pro-inflammatory cytokines and each BV-associated species with increased pro-inflammatory cytokines. Remarkably, the in vitro anti-HIV activity of CVLs from BV-positive women was stronger than that of BV-negative women. In conclusion, we found significant associations of factors, including vaginal microbiota, which can influence immune mediators in the vaginal environment in sexually active women. These factors need to be considered when establishing normative levels or pathogenic cut-offs of biomarkers of inflammation and associated risks in African women.