Growing evidence supports a link between gut microbiome dysbiosis and adverse mental states such as depression, anxiety, and other stress-related conditions. However, much less is known about how the gut microbiome relates to positive emotional well-being. Understanding this relationship could help explain the well-established connection between emotional well-being and health and provide further support for the gut-brain axis. Previous studies have been limited by small sample sizes and low-resolution microbiome profiling methods. Here, we analyzed whole-metagenome shotgun sequencing data from 2,019 stool samples collected from women in the Nurses’ Health Study II between 2019 and 2022 to investigate associations between the gut microbiome and optimism. Optimism was measured in 2017 using the validated Life Orientation Test–Revised (LOT-R). We first evaluated overall microbial diversity and community composition across optimism levels and then assessed associations of microbial species and pathways with optimism while adjusting for technical factors and relevant host covariates. We observed a modest increase in gut microbial alpha diversity with higher optimism levels. After correction for technical batch effects, overall gut microbiome composition remained significantly associated with optimism. The gut microbiome composition was also strongly influenced by host factors, including body mass index, diet, and chronic disease status. After adjustment for these host factors, 22 microbial species and 16 microbial pathways were associated with optimism at an exploratory false discovery threshold (adjusted p-values < 0.2). Several of these associations were biologically notable and supported by recent literature. For example, microbes associated with higher optimism were enriched in progestin-related functions and in pathways involved in queuosine and UMP biosynthesis. These findings suggest that gut microbial metabolism may represent an underappreciated mechanism linking the gut microbiome to optimism. This work provides a large-scale, population-based framework for studying positive emotional well-being through the lens of metagenomics and may help guide future mechanistic studies.