Aims: To determine the effects of sodium bisulfate (SBS) on the bacterial populations in cattle waste. Methods and Results: We applied SBS at 0, 60, 70 or 100 kg week(-1) to cattle waste as it accumulated on the floors of four cattle pens, housing eight cattle each. We observed significant pH decreases in all of the treated wastes on day one; however, the 60 kg week(-1) treatment returned to control levels by day four, while the others remained significantly lower. Heterotrophic plate counts of the waste revealed that all treatments reduced the bacterial populations in the wastes on day one; however, all returned to control levels by day four. The 16S rRNA gene libraries derived from the wastes revealed significant reductions in sequences associated with the phyla Bacteroidetes and Firmicutes and increases in the Proteobacteria, Actinobacteria and Spirochaetes on day one, but resembled the control by day seven. Sequences associated with Escherichia coli increased significantly after SBS application, but became undetectable by day seven. Conclusions: SBS application significantly alters the bacterial population structure of waste during the first few days of application, but the populations return to almost normal after 7 days. Significance and Impact of the Study: Application of SBS to animal waste can reduce emissions; however, biosecurity precautions must be rigorously maintained during the initial application to ensure that pathogenic E. coli is not released into the environment.