The evolutionary processes that underlie variation in plant genome size have been much debated. Abiotic factors are thought to have played an important role, with negative and positive correlations between genome size and seasonal or stressful climatic conditions being reported in several systems. In turn, variation in genome size may influence plant traits which affect interactions with other organisms, such as herbivores. The mechanisms underlying evolutionary linkages between plant genome size and biotic and abiotic factors nonetheless remain poorly understod. To address this gap, we conducted phylogenetically controlled analyses testing for associations between genome size, climatic variables, plant traits (defenses and nutrients), and herbivory across 29 oak (Quercus) species. Genome size is significantly associated with both temperature and precipitation seasonality, whereby oak species growing in climates with lower and less variable temperatures but more variable rainfall had larger genomes. In addition, we found a negative association between genome size and leaf nutrient concentration (found to be the main predictor of herbivory), which in turn led to an indirect effect on herbivory. A follow-up test suggested that the association between genome size and leaf nutrients influencing herbivory was mediated by variation in plant growth, whereby species with larger genomes have slower growth rates, which in turn are correlated with lower nutrients. Collectively, these findings reveal novel associations between plant genome size and biotic and abiotic factors that may influence life history evolution and ecological dynamics in this widespread tree genus.