Rice, supplemented withDendrobium officinale, was subjected to cofermentation usingSaccharomyces cerevisiaeFBKL2.8022 (Sc) andWickerhamomyces anomalusFBKL2.8023 (Wa). The alcohol content was determined with a biosensor, total sugars with the phenol–sulfuric acid method, reducing sugars with the DNS method, total acids and total phenols with the colorimetric method, and metabolites were analyzed using LC-MS/MS combined with multivariate statistics, while metabolic pathways were constructed using metaboAnalyst 5.0. It was found that the quality of rice wine was higher with the addition ofD. officinale. A total of 127 major active substances, mainly phenols, flavonoids, terpenoids, alkaloids, and phenylpropanoids, were identified. Among them, 26 substances might have been mainly metabolized by the mixed-yeasts fermentation itself, and 10 substances might have originated either fromD. officinaleitself or from microbial metabolism on the newly supplemented substrate. In addition, significant differences in metabolite could be attributed to amino acid metabolic pathways, such as phenylalanine metabolism and alanine, aspartate, and glutamate metabolism. The characteristic microbial metabolism ofD. officinaleproduces metabolites, which are α-dihydroartemisinin, alantolactone, neohesperidin dihydrochalcone, and occidentoside. This study showed that mixed-yeasts cofermentation and fermentation withD. officinaleboth could increase the content of active substances in rice wine and significantly improve the quality of rice wine. The results of this study provide a reference for the mixed fermentation of brewer’s yeast and non-yeast yeasts in rice wine brewing.