In situ management of soil-borne plant pathogens at various soil microsites during pre-season and in-crop is key for the successful management of diseases such as Rhizoctonia bare patch in cereal crops and Verticillium wilt in cotton. Biological suppression is considered a sustainable way of managing such diseases, but a better understanding of factors driving suppressive activities is necessary. In a suppressive soil, pathogen growth and disease incidence are repressed due to the biological activities in soil. In contrast, conducive soils do not offer the biological suppression hence disease impacts are more pronounced. During pathogen suppression, soils become inhospitable to the pathogen itself, however in the disease incidence suppression, pathogens are present but do not cause disease due to pathogen-microbiome interactions and/or from changes in a plant’s resistance to pathogens. Therefore, investigation of factors driving the suppressive activities in soil is pivotal to promote disease suppression capacities of agricultural soils. In this study, we amended the soil with carbon sources (chitin, sucrose), crops residues (wheat, sorghum and broccoli) and N-based fertilizer in short-term soil-based (7 days) and plant-based (4 weeks) bioassays under controlled environmental conditions. We investigated the responses in pathogen abundance and microbial catabolic potential. Furthermore, changes in the diversity and composition of soil microbiomes through amplicon and metagenome sequencing were also measured. Pathogen growth and disease expression responses of R. solani AG and V. dahlia were recorded in suppressive and conducive soils for wheat and cotton, respectively. We found different responses by pathogens against these amendments in soils with diverse physiochemical and suppression properties. For example, the addition of a simple carbon source (e.g., sucrose) reduced R. solani AG8 growth in all soil however, V. dahlia growth varied between suppressive and conducive soils. Similarly, the response by the two pathogens to the addition of chitin was different as well as differences in the total fungi. While N-fertilizer enhanced V. dahlia growth, crop residue amendments result in the decline of total fungi growth. Similarly, multivariate analysis of the genetic diversity of bacteria, fungi and functional groups showed significant differences between different amendments and soil types. For example, the addition of sorghum, broccoli and chitin increased the relative abundances of soil fungi and bacteria with antibiosis, antifungal and plant growth promoting capabilities. Overall, these results suggest that understanding the intricacies of complex microbiome-pathogen interactions is important to identify amendments that can enhanced suppressive activities in agricultural soils.