Background Managed southern white rhinoceros (Ceratotherium simum simum) serve as assurance populations for wild conspecifics threatened by poaching and other anthropocentric effects, though many managed populations experience subfertility and reproductive failure. Gut microbiome and host health are inextricably linked, and reproductive outcomes in managed southern white rhinoceros may be mediated in part by their diet and gut microbial diversity. Thus, understanding microbial dynamics within managed populations may help improve conservation efforts. We characterized the taxonomic composition of the gut microbiome in the managed population of female southern white rhinoceros (n = 8) at the North Carolina Zoo and investigated the effects of seasonality (summer vs. winter) and age classes (juveniles (n = 2; 0–2 years), subadults (n = 2; 3–7 years), and adults (n = 4; >7 years)) on microbial richness and community structure. Collection of a fecal sample was attempted for each individual once per month from July-September 2020 and January-March 2021 resulting in a total of 41 samples analyzed. Microbial DNA was extracted and sequenced using the V3-V4 region of the 16S rRNA bacterial gene. Total operational taxonomic units (OTUs), alpha diversity (species richness, Shannon diversity), and beta diversity (Bray-Curtis dissimilarity, linear discriminant analysis effect size) indices were examined, and differentially enriched taxa were identified. Results There were differences (p < 0.05) in alpha and beta diversity indices across individuals, age groups, and sampling months. Subadult females had higher levels of Shannon diversity (Wilcoxon, p < 0.05) compared to adult females and harbored a community cluster distinct from both juveniles and adults. Samples collected during winter months (January-March 2021) possessed higher species richness and statistically distinct communities compared to summer months (July-September 2020) (PERMANOVA, p < 0.05). Reproductively active (n = 2) and currently nonreproductive adult females (n = 2) harbored differentially enriched taxa, with the gut microbiome of nonreproductive females significantly enriched (p = 0.001) in unclassified members of Mobiluncus, a genus which possesses species associated with poor reproductive outcomes in other animal species when identified in the cervicovaginal microbiome. Conclusion Together, our results increase the understanding of age and season related microbial variation in southern white rhinoceros at the North Carolina Zoo and have identified a potential microbial biomarker for reproductive concern within managed female southern white rhinoceros.