Acanthamoeba keratitis (AK) is a severe infection linked to orthokeratology lens use, whereas the involvement of conjunctival microbiota in AK remains poorly understood. This study investigates microbiota dysbiosis in AK pathogenesis to inform microbiota-based interventions.Conjunctival swabs from 14 patients with AK and 10 healthy controls underwent 16S rRNA sequencing. Microbiome analysis compared diversity, taxa, and metabolic pathways. Functional assays quantified Achromobacter-enhanced Acanthamoeba adhesion and migration. Metagenomics and fluorescence in situ hybridization (FISH) with species-specific probes confirmed endosymbiosis.Patients with AK showed reduced bacterial diversity compared with the healthy controls (P < 0.001) but similar richness. Relative abundance of Achromobacter in the AK group was higher compared to the healthy control group (P < 0.001). Achromobacter dominated microbiota among the AK group, being identified as a key biomarker via the linear discriminant analysis effect size (LEfSe). In vitro, Achromobacter increased Acanthamoeba adhesion (P = 0.007) and the migration area (P < 0.05). Metagenomic analysis and FISH further showed Achromobacter spp. as potential endosymbionts of Acanthamoeba. Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed upregulated phenylalanine, fatty acid, and propanoate metabolism in the AK group (all P < 0.001). MetaCyc highlighted enriched pyruvate fermentation to isobutanol, aerobic respiration I, and L-isoleucine biosynthesis II in the AK group (P < 0.001).AK-associated conjunctival dysbiosis features Achromobacter dominance, reduced diversity, and altered metabolism. Achromobacter is associated with enhanced adhesion and migration of Acanthamoeba, indicating a possible symbiotic interaction and its potential as a biomarker and therapeutic target.