Marine spatial planning (MSP) is designed to divide the sea area into different types of functional zones, to implement corresponding development activities. However, the long-term impacts of anthropogenic activities associated with MSP practice on the marine microbial biosphere are still unclear. Yalu River Estuary, a coastal region in northeast of China, has been divided into fishery & agricultural (F&A) zone, shipping & port (S&P) zone and marine protected area (MPA) zone by a local MSP guideline that has been run for decades. To examine the effects of long-term executed MSP, benthic bacterial communities from different MSP zones were obtained and compared in this study. The results revealed significant differences in the bacterial community structure and predict functions among different zones. Bacterial genera enriched in different zones were identified, including SBR1031 in MPA, Woeseia and Sva0996 in S&P, and Halioglobus in F&A. In addition, correlations between some bacterial genera and sediment pollutants were uncovered. Furthermore, bacteria related to sulphide production were more abundant in the F&A zone, which was according to the accumulation of sulphides in this area. Moreover, bacteria associated with chemoheterotrophy and fermentation were more predominant in the S&P zone, consistent with high levels of organic matter and petroleum caused by shipping. Our findings indicated benthic bacterial communities could bring to light the anthropogenic activity footprints by different activities induced by long-term MSP practice.