Denture stomatitis (DS) is an inflammatory condition that affect denture wearers and is characterized by erythema of the mucosa opposing the denture. DS is often associated with oral microbiome dysbiosis. We used shotgun metagenomics to investigate the association between the denture-associated oral microbiome (DAOM) and DS in older adults living in long-term care facilities. We included participants with DS (n = 28) and age-and sex-matched removable denture wearers without signs of DS (n = 28). Clinical oral examinations were performed, and demographic and medical data were obtained from medical records. Median (interquartile range) age of participants was 88 (9) years; 75% were females. Beta diversity differed between the DS and non-DS groups (Bray-Curtis dissimilarity, p = 0.01; Jaccard index, p = 0.004). Two phyla, nine genera, and 15 species differed significantly between groups, with the genera Candida and Scardovia, and species Candida albicans, Aggregatibacter actinomycetemcomitans, and Scardovia inopinata being enriched in DS. Network analysis revealed strongly interconnected microbial communities and more prominent bacterial-fungal co-occurrence in DS than in non-DS. These findings indicate that DS is associated with significant alterations in the DAOM, which may contribute to inflammation. Microbiome-targeted strategies are needed for the management of DS.