A thorough exploration of Ecosystem Services (ESs) and their intricate interactions across time and space is a prerequisite for the sustainable management of multiple ESs. This study aimed to systematically evaluate the ESs of the middle reaches of the Yangtze River Urban Agglomeration (MRYRUA) across multiple spatial and temporal scales, thereby enhancing ecosystem management and informed scientific decision-making. Specifically, this study employed the InVEST model, hot spot analysis, a geographically weighted regression model, and self-organizing feature mapping combined with K-means clustering to systematically quantify the spatiotemporal characteristics, trade-offs, synergies, and ecosystem service clusters of habitat quality (HQ), water yield (WY), carbon storage (CS), soil conservation (SC), and landscape aesthetics (LA) at grid and county scales from 2000 to 2020. The results revealed the following: (1) There was significant spatial heterogeneity among various ESs, with an overall spatial pattern exhibiting layered and interwoven variations. (2) Trade-offs predominantly characterized the relationships among ESs in the MRYRUA, with the absolute values of correlation coefficients mostly reaching their nadir in 2010. The interaction strengths between HQ and CS, and between CS and SC, increased with scale, while the relationships and strengths between LA and other ESs were less affected by scale changes. (3) At the grid scale, five types of ecosystem service bundles (ESBs) were identified, whereas at the district scale, four types of ESBs were delineated, including three common types: the WY–LA synergy bundle, Ecological transition bundle, and Key synergetic bundle, and three distinct types: the HQ–CS synergy bundle, Integrated ecological bundle, and Key synergetic bundle. The transitions of these ESBs over the 20 year period generally exhibited fluctuating evolutionary characteristics, with more pronounced fluctuations as the scale expanded. The results improve our comprehension of how ESs are related across various scales and provide theoretical and scientific references for multi-scale sustainable ecosystem zoning management and ecological environment governance.