This study compared the safety of a new tampon with a four-winged apertured film cover over its nonwoven cover to improve leakage performance with that of a commercial tampon with a nonwoven cover only. Healthy women (evaluable, n = 69) were randomized to crossover between test and reference tampons in two consecutive menstrual cycles. Qualitative and quantitative analyses of vaginal cultures were conducted pre-, mid-, and postmenstrually for a broad panel of microorganisms, and colposcopy was performed. Similar to previous studies, prevalence and mean colony counts of the majority of microorganisms generally increased midmenstrually and returned or began to return postmenstrually. In contrast to most previous studies, Lactobacillus species remained at similar levels throughout the cycles with both tampons. Neither tampon was associated with clinically significant microbiological changes or abnormalities or with vaginal/cervical epithelial integrity changes on colposcopy. Microbiological and colposcopic evaluations indicate that the apertured film-covered tampon is safe.