Chlorothalonil is a nonsystemic fungicide, and it is one of the most widely detected pesticides in bee hives. The effect of chlorothalonil on the survival, weight, and gut microbiota of immature Apis mellifera L. reared in vitro was studied. Larvae were fed 1, 2, 4, 8, and 16 μg/mL chlorothalonil and compared with larvae fed the negative control (diet without any additives), positive control (45 mg/L dimethoate), and solvent control (2% acetone). Compared with the control groups, the survival of the 2, 4, 8, and 16 μg/mL chlorothalonil treatments was significantly reduced. The no-observed-adverse-effect concentration of chlorothalonil was 1μg/mL. Chlorothalonil had no significant effect on larval weight. The gut bacterial community composition of newly emerged bees was determined by PacBio 16S rDNA gene sequencing. linear discriminant analysis effect size (LEFSe) analysis showed that Pseudomonadales and Burkholderiales were affected by exposure to chlorothalonil. Chlorothalonil reduced the survival of honey bee larvae and altered the gut microbiota of newly emerged bees. The risk of pesticides to honey bees is related to their toxicity and exposure dose.