Purpose The discovery of complete ammonia oxidizers (comammox) has revolutionized the traditional understanding of two-step nitrification. However, little is known about the ecological niches and environmental driving factors of comammox Nitrospira in acidic tea plantation ecosystems. Methods We investigated the occurrence and community composition of comammox Nitrospira in a typical acidic tea plantation soil in Anji County, China. This was achieved through a combination of microcosm incubation, addition of specific nitrification inhibitors, molecular biology assays, phylogenetic analysis based on functional gene sequences, and statistical correlation with soil chemical properties. We compared comammox Nitrospira with ammonia-oxidizing archaea (AOA) and bacteria (AOB) and evaluated the combined or individual impacts of soil pH gradients and/or chlorate and 3,4-dimethylpyrazole phosphate (DMPP). Results Quantitative analysis showed that AOA were the dominant ammonia oxidizers. Comammox Nitrospira were detected in all tested soils across three pH gradients, exhibiting greater gene abundance than AOB, and were significantly reduced by both chlorate and DMPP. Clade A2 comammox Nitrospira comprised the majority groups, with operational taxonomic units (OTUs) 547, 526, and 521 emerging as the dominant floras. The abundance of comammox Nitrospira was mainly affected by pH and ammonium concentrations. AOB abundance, nitrate contents, and AOA abundance were identified as vital factors affecting the comammox Nitrospira community under control, chlorate-added, and DMPP-added conditions, respectively. Conclusions This study demonstrated the presence and biodiversity of comammox Nitrospira in tea plantation soils with different pH gradients, indicating that comammox Nitrospira may play a significant and previously underestimated role in microbial nitrification in strongly acidic tea soils.