Vaginal practices may interfere with the use and/or the effectiveness of female-initiated prevention methods. We investigated whether vaginal practices differed by randomization group in a phase III trial of the diaphragm with lubricant gel (MIRA) in Sub-Saharan Africa (n = 4925), and if they were associated with consistent use of study methods. At baseline, vaginal practices were commonly reported: vaginal washing (82.77%), wiping (56.47%) and insertion of dry or absorbent materials (20.58%). All three practices decreased during the trial. However, women in the intervention group were significantly more likely to report washing or wiping during follow-up compared to those in the control group. Additionally, washing, wiping, and insertion, were all independently and inversely associated with consistent diaphragm and gel use and with condom use as well, regardless of study arm. A better understanding of the socio-cultural context in which these practices are embedded could improve educational strategies to address these potentially modifiable behaviors, and may benefit future HIV prevention interventions of vaginal methods.