The accuracy and suitability of use of a single intravaginal swab (SIS) for polymerase chain reaction detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, and human papillomavirus infection was assessed in a cross-sectional study of 841 active-duty military women. The SIS, compared with standard diagnostic tests, allowed detection of more gonorrhea, more chlamydial infection, and more trichomoniasis. Sensitivity and specificity of SIS detection compared with adjudicated true-positive diagnoses were 95.8% and 97.8%, respectively, for gonorrhea, 94.6% and 99.3% for chlamydial infection, and 92.2% and 98.2% for trichomonal infection. Results with SISs were comparable to those with cervical swabs tested for human papillomavirus. Assay of clinician-collected and self-collected SISs yielded prevalences similar to those of standard diagnostic tests for all sexually transmitted infections. Therefore, the use of SISs is acceptable for the simultaneous diagnosis of multiple sexually transmitted infections and has potential for use as a self-administered diagnostic tool with widespread applicability among women.