Manure applications can benefit crop productivity by adding required nutrients and organic matter to soil. There is a paucity of information on how soil microbial community dynamics will be altered by the application of manure to different landscape positions. Thus, an in situ field study was conducted during the summer and winter months to evaluate microbiological properties of three soil types that have evolved because of different landscape positions in an agricultural field. The three Coastal Plain soils investigated were Bama (sandy loam), Lynchburg (loam), and Goldsboro (loam) representing the landscape position of a summit, drainageway, and sideslope, respectively. Composted dairy manure was incorporated into in situ soil cores at a rate of 350 kg N ha−1 and compared with unamended controls. Soil microbial biomass N and dehydrogenase enzyme activity were determined to evaluate changes in the microbial biomass size and activity, whereas phospholipid fatty acid analysis was used as an indicator of the microbial community structure. Addition of composted dairy manure increased microbial activity and N immobilization, representing a shift in microbial response resulting from changes in substrate availability. This was most evident during summer months, with the composted dairy manure increasing dehydrogenase enzyme activity 21% and microbial activity 20% compared with without manure, suggesting that seasonal timing of application will influence microbial activity. Microbial properties were also impacted by landscape position. The drainageway landscape position soil, a loam, had the highest microbial biomass and microbial activity. Changes in microbial community structure using phospholipid fatty acid profiles were evaluated with canonical discriminate analysis. This analysis indicated that a shift in microbial community structure occurred between season, manure application, and landscape position. Findings from this study suggest that changes in soil variability from landscape positions and season can impact the growth and dynamics of the microbial community when manure is applied to agricultural fields.