Technical Abstract: Polyacrylamide (PAM, anionic formulation of molecular size 12 MDa and 35% charge density) has been reported to increase aggregation and improve soil physical properties in United States southeastern Coastal Plain loamy sand soils, but nothing is known about the effects of PAM on microbes associated with soil aggregation. The aims of this study were to: 1) isolate and identify the predominant, heterotrophic bacteria from the cultural portion of the microaggregates (0.25-0.05 mm) of soils treated with PAM and/or organic matter (OM), 2) test the ability of the isolates to aggregate soil in vitro, and 3) quantify the amount of soil aggregating basidiomycete fungi in the different aggregates size fractions generated from the treated soils. Pots containing a Norfolk soil (a blend of 90% E horizon and 10% Ap horizon), PAM (0, 30 and 120 mg kg-1) and OM (wheat [(Triticum aestivum)] and pecan [Carya illinoinensis]) were incubated for 96 days at 10% water content. Fatty Acid Methyl Ester (FAME) profiling was used to identify the predominant bacteria species from all the treatments, and DNA sequencing was used to find their position in a phylogeny with known taxa. Among all the identified species, 19 were soil aggregators as demonstrated by an in vitro soil sedimentation assay. When comparing the proportion of soil aggregating isolates between treatments, there was a PAM effect across all residue types indicating that the highest proportion was with 120 mg kg-1 PAM. This suggests that soil conditions created by addition of 120 mg kg-1 PAM favored the growth of bacteria functioning as soil aggregators. There was evidence of interaction between PAM and residue type. Differences were found in the PAM effect but only for no residue added. Enzyme-linked immunosorbent assay (ELISA) indicated a significantly higher amount of soil aggregating basidiomycete fungi in macroaggregate size classes of 2.00-1.00 and 1.00-0.50 mm generated from PAM and/or wheat residue amended-soil compared to the control soil, whereas there was no difference detected in treatments with pecan residue added, suggesting that PAM and PAM with wheat can be utilized as a source of nutrient for the basidiomycetes but pecan appeared to inhibit their growth. This study is the first to provide evidence that adding anionic PAM to soil favored the growth and survival of specific fungi and bacterial species functioning as soil aggregators in vitro.