The effect of acidifiers on nitrogen transformation during manure wastewater storage remains unclear. In this study, we investigate the effects of two types of acidifiers on nitrogen transformation, bacterial structure, and functional genes during manure wastewater storage. The results indicate that the internal reactions in manure wastewater storage could be divided into three stages, i.e., nitrification (first 35 days), denitrification (mainly from days 36 to 84), and the final stage during which the internal environment of the manure wastewater stabilized. The addition of acidifiers effectively reduced nitrogen loss, with the total nitrogen content increasing from 12.8 to 25.83%, and ammonia emissions decreasing from 31.67 to 43.81%. The optimal nitrogen retention effect was observed for sulfuric acid. Furthermore, acidifiers primarily inhibited microbial activity and the abundance of functional genes, without altering the dominant bacterial community structure or nitrogen transformation pathways. These results provide a foundation for the use of acidifiers to reduce nitrogen loss during manure wastewater storage.