Leaf-cutting ants represent a significant threat to forests and agriculture. In this context, the use of plant-derived substances has garnered growing interest as an alternative to synthetic insecticides. This study aimed to evaluate the effects of different concentrations of diphenyl disulfide and lyral, compounds present in the essential oil of Tropaeolum majus (Brassicales: Tropaeolaceae), on the leaf-cutting ant Atta sexdens and its symbiotic fungus Leucoagaricus gongylophorus. First, we assessed the effects of these compounds on fungal growth. Subsequently, we evaluated the impact of topical application and exposure to the compounds through an artificial diet on the survival of gardener and forager worker ants. We also analyzed worker ant interactions using a software tool for behavioral assessments, to evaluate the spread of the compounds via topical application. No significant changes were observed in the mass or growth area of fungi exposed to diphenyl disulfide or lyral. Through topical application, diphenyl disulfide demonstrated a toxic effect that increased with higher concentrations of the compound, while lyral showed no toxic effect on any ant caste. No significant effects on ant mortality were observed when the compounds were administered via artificial diet, nor was there inhibition of ant feeding, indicating that the compounds did not directly influence food consumption. Diphenyl disulfide increased interactions between treated and untreated ants compared to lyral and the control. However, this increase in interactions did not compromise colony activities. Thus, the results suggest that diphenyl disulfide is a compound with characteristics worth exploring to assess its potential for managing Atta sexdens.