Background: Bacterial vaginosis is associated with sexual activity, but mechanisms for this association are unclear. Study Design: Cross-sectional analysis of data from women reporting sex with women who provided information on sexual behaviors as part of a study of vaginal bacteria was conducted. Vaginal bacteria were detected by semiquantitative culture. Binomial regression with log-link evaluated associations between detection of bacteria and sexual behaviors reported to occur before enrollment. Linear regression evaluated associations between these behaviors and quantity of bacteria. Results: Of the 320 women, 216 (68%) were colonized with H2O2-producing lactobacilli and 142 (44%) with Gardnerella vaginalis. Colonization with G. vaginalis was associated with >20 digital-vaginal sex acts (risk ratio, 2.01; 1.22, 3.29) or >10 toy-vaginal acts in the past 3 months (risk ratio, 1.76; 1.32, 2.36). Quantity of H2O2-producing lactobacilli was 1.3 log lower in colonized women reporting >10 acts of insertive vaginal sex-toy use in the past 3 months (95% confidence interval:-2.04,-0.56), 1.19 log lower with toy-vaginal sex in past 7 days (-1.75,-0.62), and 0.78 log lower in women sharing toys with a partner (-1.25,-0.31). Conclusions: Vaginal insertive use and sharing of sex toys were associated with decreased quantities of H2O2-producing lactobacilli and higher risk of colonization with G. vaginalis. © 2011 American Sexually Transmitted Diseases Association All rights reserved.