Aim: To evaluate the effect of long-term annual land applications of Class B biosolids on soil bacterial diversity at University of Arizona Marana Agricultural Field Center, Tucson, Arizona. Methods and Results: Following the final of 20 consecutive years of application of Class B biosolids in March 2005, followed by cotton growth from April to November 2005 surface soil samples (0-30 cm) were collected from control (unamended) and biosolid-amended plots. Total bacterial community DNA was extracted, amplified using 16S rRNA primers, cloned, and sequenced. All 16S rRNA sequences were identified by 16S rRNA sequence analysis and comparison to known sequences in GenBank (NCBI BlastN and Ribosomal Database Project II, RDP). Results showed that the number of known genera (identifiable > 96%) increased in the high rate biosolid plots compared to control plots. Biosolids-amended soils had a broad phylogenetic diversity comprising more than four major phyla: Proteobacteria (32%), Acidobacteria (21%), Actinobacteria (16%), Firmicutes (7%), and Bacteroidetes (6%) which were typical to bacterial diversity found in the unamended arid southwestern soils. Conclusion: Bacterial diversity was either enhanced or was not negatively impacted following 20 years of land application of Class B biosolids. Significance and Impact of the Study: This study illustrates that long-term land application of biosolids to arid southwestern desert soils has no deleterious effect on soil microbial diversity.