Background: Vaginal douching is consistently associated with bacterial vaginosis (BV), but whether it is a cause or result of BV remains unknown. The association between BV and other feminine hygienic behaviors is less studied; if BV symptoms caused behavior change then all hygiene behaviors might be more common among women with BV. Lack of association between nondouching hygiene behavior and BV would argue against reverse causation. Methods: In the Longitudinal Study of Vaginal Flora 3620 women had 13,517 visits where BV (Nugent score) was assessed. Associations between hygienic behavior and BV were assessed by Poisson regression. Results: After adjusting for demographic and sexual behavior factors, neither type of underwear (nylon vs. cotton prevalence ratio (PR) 1.05, 95% CI: 0.97-1.13), menstrual protection (tampons vs. pads; PR: 1.04, 95% CI: 0.95-1.12; pads and tampons vs. pads 1.00, 95% CI: 0.92-1.07), use of pads or panty liners when not menstruating (PR: 0.99, 95% CI: 0.95-1.05), nor weekly or greater use of hygiene spray (PR: 1.01, 95% CI: 0.94-1.09), powder (PR: 1.02, 95% CI: 0.96-1.07) or towlettes (PR: 1.03, 95% CI: 0.94-1.13) were strongly associated with BV. PR for daily versus less than daily bathing and showering were 1.06 (95% CI: 1.02-1.12) and 1.04 (95% CI: 1.00-1.09). Douching remained associated with BV (PR for weekly or greater vs. never 1.17, 95% CI: 1.09-1.26) and was not substantially impacted by adjustment for other hygienic behavior. Conclusions: Douching, but not other feminine hygiene behaviors, is significantly associated with BV, providing additional evidence that douching may be causally associated with BV and is not simply a response to BV symptoms. © Copyright 2010 American Sexually Transmitted Diseases Association.