The intense use of pesticides in agricultural activities for the last several decades has caused contamination of the ecosystems connected with crop fields. Despite the well-documented occurrence of pesticide biodegradation by microbes, natural attenuation of atrazine (ATZ), and its effects on ecological processes in subtropical forested areas, such as Iguaçu National Park located in Brazil, has been poorly investigated. Subtropical environments sustain a great degree of fungal biodiversity, and the patterns and roles of these organisms should be better understood. This work aimed to evaluate nine ligninolytic-producer fungi isolated from the INP edge to degrade and detoxify ATZ solutions. ATZ degradation and the main metabolites produced, including deisopropylatrazine and deethylatrazine (DEA), were analyzed using dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometer. Four fungi were able to degrade ATZ to DEA, and the other five showed potential to grow and facilitate ATZ biodegradation. Furthermore, two strains of Fusarium spp. showed an enhanced potential for detoxification according to the Allium cepa (onion) test. Although the isolates produced ligninolytic enzymes, no ligninolytic activity was observed in the biodegradation of ATZ, a feature with ecological significance. In conclusion, Ascomycota fungi from the INP edge can degrade and detoxify ATZ in solution. Increasing the knowledge of biodiversity in subtropical protected areas, such as ecosystem services provided by microbes, enhances ecosystem conservation.