Understanding the role of root microbiota is crucial in sustainable forest management but remains challenging, especially for tropical trees. We developed an efficient and low-toxicity method to extract and amplify the fungal DNA associated with Aucoumea klaineana Pierre fine roots. To improve DNA quality, we optimized a commercial extraction kit by incorporating activated charcoal and modifying incubation periods. This enhanced protocol, combined with bovine serum albumin during PCR, effectively mitigated inhibitors present in A. klaineana tree root samples. This approach opens new perspectives for studying the microbiota of tropical trees.