Applying manure or municipal waste biosolids through landspreading may alter soil microbial community diversity and function especially if antimicrobial chemicals like chlortetracycline (CTC), administered in livestock feed to promote animal growth and health, or tetracycline (TET), utilized in human health, are present. Herbicide degradation rates may decrease as microbial diversity changes. In this study, soil microbial diversity and community shifts were examined using several techniques including most probable number (MPN) and denaturing gradient gel electrophoresis (DGGE) of the V3 region of the 16S rRNA gene pool at 0, 7, 28, and 42 days after treatment (DAT). Treatments included 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide applied to soil treated with manure from swine fed untreated or CTC containing rations and municipal waste biosolids either untreated or spiked with TET. Colony forming units (CFU) of heterotrophic microorganisms culturable under aerobic conditions on R2A agar plates were 10-fold higher 7DAT in soil treated with manure containing CTC compared with all other treatments but it is unclear if the increase in CFU was due to native soil or manure organisms. The density of 2,4-D degrading microorganisms using the MPN method indicate a nearly 20 fold increase in number of 2,4-D degraders after adding 2,4-D to soil samples compared to soil samples without the application. The addition of manure or biosolids with or without antibiotics did not influence the numbers of 2,4-D degrading organisms. DGGE analysis allowed for representation of all microorganisms present, culturable or not. Significant shifts in the 16S rRNA gene pool occurred within and among the different treatments over time.