Citrus is one of the most widely consumed fruits in the world, and its cultivation industry continues to develop rapidly. However, the roles of soil protistan communities during citrus growth are not yet fully understood, despite the potential significance of these communities to the health and quality of citrus. In this study, we examined the soil properties and protistan communities in Eureka lemon farmlands located in Chongqing, China, during the flowering and fruiting stages of cultivation, both in greenhouse and open-field settings. In general, the majority of the measured soil properties (including nutrients and enzyme activities) exhibited higher values in open-field farmlands in comparison to those observed in greenhouse counterparts. According to the results of high-throughput sequencing based on the V9 region of eukaryotic 18S rRNA gene, the diversity of soil protistan communities was also higher in open-field farmlands, and both lemon growth stage and cultivation modes showed significant effects on soil protistan compositions. The transition from traditional agricultural practices to greenhouse farming resulted in a significant transformation of the soil protistan community. This transformation manifested as a shift towards a state characterized by diminished nutrient cycling capabilities. This decline was evidenced by an increase in phototrophs (Archaeplastida) and a concomitant decrease in consumers (Stramenopiles and Alveolata). Community assembly analysis revealed deterministic processes that controlled the succession of soil protistan communities in lemon farmlands. It has been established that environmental associations have the capacity to recognize nitrogen in soils, thereby providing a deterministic selection process for protistan community assembly. Furthermore, a production index was calculated based on 12 quality parameters of lemons, and the results indicated that lemons from greenhouse farms exhibited a lower quality compared to those from open fields. The structure equation model revealed a direct correlation between the quality of lemons and the cultivation methods employed, as well as the composition of soil protists. The present study offers insights into the mechanisms underlying the correlations between the soil protistan community and lemon quality in response to changes in the cultivation modes.