Polluting enterprises represent a conflict between ecological protection and high-quality development in the Yellow River Basin. Optimizing and regulating the spatial layout of polluting enterprises is an important measure to promote the coordinated development of ecology and economy in the Yellow River Basin. By constructing a spatial point database of the polluting enterprises, and using multiple spatial data analysis such as the standard deviation ellipse, kernel density analysis, Ripley's K function and the global spatial autocorrelation, this study examined the directional trend, spatial agglomeration form, the degree of spatial agglomeration at different distance scales, and spatial correlation of polluting enterprises in the Yellow River Basin from 1979 to 2020. This study further examined the spatial association of polluting enterprises with the natural environment, economic environment, and other geographical environments using Geodetector for factor detection and interactive detection. The results showed that: (1) From 1979 to 2020, the number of polluting enterprises in the Yellow River Basin continued to grow. Polluting enterprises were closely clustered around the main channel and tributaries of the Yellow River, with the spatial distribution range of such enterprises expanding toward the source of the Yellow River. (2) The polluting enterprises exhibited significantly spatial agglomeration in the Yellow River Basin. As the degree of agglomeration of polluting enterprises increased, the spatial scope of their agglomeration distribution expanded, and generally presented four spatial agglomeration forms: central agglomeration, central agglomeration with multi-point dispersion, central continuous agglomeration with multi-point dispersion, and central continuous agglomeration. The agglomeration degree of polluting enterprises initially increased, and then decreased, as the geographical distance scale increased. Furthermore, the spatial distribution range of the polluting enterprises with the highest degree of agglomeration was expanding. (3) Economic factors were the dominant drivers of the spatial distribution of polluting enterprises in the basin, while the interactions between social factors and other factors had a strong impact on the distribution. The spatial correlation type of polluting enterprises and the differences in economic and social development in the basin had a significant club convergence effect. The polluting enterprises had significant positive spatial correlation, and their spatial correlation types were mainly high-high agglomeration and low-low agglomeration. The high-high agglomeration type was mainly distributed in the lower reaches of the Yellow River Basin, and the low-low agglomeration type was mainly distributed in the upper and middle reaches of the Yellow River Basin. This study provides support for the spatial layout optimization and regulation of polluting enterprises in the Yellow River Basin.