Maximizing the role of ecosystem services in promoting household well-being, as well as ensuring ecological function positioning, is the key to achieving sustainable development in karst regions. Current studies on the effect of ecosystem services on household well-being mainly focus on either macro or micro single scales, with limited attention given to their cross-scale associative effects, causing a one-sided understanding of the complex impact mechanism on household well-being and the limitations in policy-making. In this study, taking a typical karst region of Huanjiang County as an example, we constructed a household-village hierarchical linear model based on estimated ecosystem services and survey data from 677 households, clarified the spatiotemporal characteristics of regional macro-level ecosystem services and household well-being, analyzed the cross-scale direct and indirect impacts of ecosystem services on household well-being, and propose targeted optimization strategies. The results indicated that (1) From 2012 to 2020, ecosystem services in Huanjiang County showed an overall increasing trend and significant spatial heterogeneity. The average score of household well-being was 3.51, with safety well-being scoring the highest at 4.30 and income well-being obtaining the lowest score at 2.68; spatially, the regions with high household well-being were distributed in the southwestern of the study area, while regions with low household well-being were concentrated in the northeastern regions. (2) Individual characteristics such as age, education level, and previous experience in a poverty-stricken household, significantly impacted household well-being. At the village level, improvements in food production, scientific education, and air purification would directly enhance household well-being, and air purification had a more pronounced role, showing the satisfaction of material needs and the pursuit of high-level needs for households. (3) Carbon sequestration showed an impact on individual characteristics such as education level and age, thereby generating a positive cross-scale correlation effect on household well-being, emphasizing the critical importance of ecological restoration policies; food production exerted an influence on age, thereby generating a negative regulatory effect on household well-being, revealing regional social issues such as insufficient young labor force and hollowing out. Compared to provisioning and cultural services, regulating services exerted a more significant cross-scale indirect effect on enhancing regional household well-being. In ecologically fragile karst regions, there should be a focus on improving critical regulating services, and effective measures need to be carefully formulated to alleviate the issue of rural depopulation and low education level, and then optimize the welfare effect of ecosystem services and achieve regional high-quality development.