The Eastern Indigo Snake (Drymarchon couperi), a federally threatened species, serves as a valuable model for examining the impacts of captivity and infection on gut microbial composition in reptiles. This study assessed the cloacal microbiome of wild and captive D. couperi using shotgun metagenomic sequencing. Samples were divided into three groups for comparative microbiome analysis: captive snakes with C. serpentis infection, captive snakes without C. serpentis infection, and wild snakes. Alpha (Shannon index, paired Wilcoxon test) and beta diversity (Bray-Curtis dissimilarity, PERMANOVA, CAP) metrics were used to assess microbial richness and community composition across groups. Furthermore, a LEfSe analysis was conducted to identify microbial taxa significantly enriched in C. serpentis–infected versus uninfected captive snakes. Bacterial, fungal, bacteriophage, nematode, and protozoan taxa were significantly enriched in C. serpentis–infected snakes compared to uninfected captive snakes, based on an LDA score > 2.5 and P < 0.05. Total taxa species richness was consistent between C. serpentis-positive and negative captive snakes (P=0.55) while wild snake samples were significantly more diverse (P=0.026). Wild snakes also exhibited a significantly increased richness of fungi (P=0.044), protozoa (P=0.012), and nematodes (P=0.008) compared to their captive counterparts. This study offers the first in-depth characterization of the cloacal microbiome in reptiles, specifically in this species, using shotgun metagenomic sequencing. The findings establish a foundation for exploring microbiota–host interactions with implications for reptile health, disease ecology, and conservation management.