The complex and enormous diversity of microbiome associated with plant roots is important for plant health and is shaped by numerous factors. This study aimed to unravel the effects of a pesticide-fertilizer combination on the rhizosphere microbiome of field-grown sugarcane. A field trial on sugarcane was conducted in Zhanjian city, Guangdong Province of China, and bulk soil and rhizosphere samples were collected 3 weeks after planting. We examined the effects of clothianidin and/or organic fertilizer treatments on the composition, diversity, and function of rhizosphere microbial communities using 16S rRNA gene and ITS1 gene amplicon sequencing. Compared with the controls (no pesticide or fertilizer used), the microbial community that resulted from treating with the pesticide-fertilizer (SPF) had a higher relative bacterial diversity and abundance, as well as contributing more comprehensive functions to sugarcane. The bacterial and fungal compositions at different taxonomic levels affected by clothianidin in the SPF and SP (with pesticide addition) were different from the effects experienced in the other treatments. With organic fertilizers added to SPF, the abundance of soil beneficial bacteria Bacillus, Paenibacillus, and Brevibacillus were highly improved, as well as the microbial function. It indicated that the SPF treatment could counteract the effects caused by mixing with clothianidin when compared to the SP treatment with only pesticide addition. Moreover, four bacterial genera including Dyella, Sphingomonas, Catenulispora, Mucilaginibacter, and Tumebacillus were significantly increased in the SPF and SP groups, which was reported to degrade clothianidin and could improve the soil health. The findings of the study provide insights into the interaction between the rhizosphere soil microbiome and a pesticide-fertilizer integration that may help improve application for pesticide-fertilizer in sugarcane fields.