Introduction and Hypothesis <p>Postmenopausal women with recurrent urinary tract infections (RUTI) are repeatedly exposed to antibiotics; therefore, they are at risk for colonization by multi-drug resistant organisms. Methenamine hippurate (MH), an antibiotic alternative, has shown clinical promise in preventing RUTI; its activity is traditionally attributed to formaldehyde production, though the precise mechanism of action remains uncertain. We hypothesize that MH administration alters the urobiomes of women with RUTI.</p> Methods <p>We conducted a longitudinal study of postmenopausal women with a clinical history of RUTI, evaluating their urobiomes for 3 months. Expanded quantitative urine culture (EQUC) assessed diversity and urobiome composition of catheterized urine, voided urine, and peri-urethral swabs; 16S rRNA gene sequencing determined taxonomic classification, beta diversity, and differential abundance analysis of voided urine only.</p> Results <p>No UTIs occurred for any participant. Instead, we observed improvement in symptoms. EQUC and 16S sequencing revealed predominance of <i>Enterobacteriaceae</i>, especially <i>E. coli</i>, and members of several Gram-positive genera, including emerging uropathogens. Following MH therapy initiation, diversity of catheterized urine specimens increased and voided urine microbiomes of most participants differed significantly during at least one post-treatment week relative to pre-treatment composition. Differential abundance analysis revealed many significantly different taxa; several were not among the most abundant.</p> Conclusions <p>MH treatment did not sterilize the bladder nor did it eliminate uropathogens. Instead, it uniquely altered each participant’s voided urine microbiome. To understand the mechanism of action, researchers should look beyond suspected uropathogens and consider the participant and the rest of their urobiome.</p>