Perfluorononanoic acid (PFNA), widely employed in surfactants, coatings, plastics, corrosion inhibitors, and fire-extinguishing agents, is less regulated than PFOS or PFOA but displays higher bioaccumulation and potential toxicity. Most toxicity assessments have focused on mammals, fish, and algae, with limited research on ground-dwelling arthropods, especially ants. Here, we examined PFNA's toxic effects on red imported fire ants (RIFAs), a prevalent ground-dwelling species in South China. Stomach and contact toxicity experiments revealed that PFNA significantly reduced RIFA viability, with poisoning severity correlating positively with both dose and exposure duration. PFNA inhibited key behaviors-including aggregation, climbing, grasping, crawling, and excavation-and impacted survival. For instance, after 12 hours at 0.12 mg/g PFNA, large and medium workers excavated only 0.136 g and 0.064 g of quartz sand, respectively, significantly less than controls. Lethal concentrations (LC