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Abstract Background Fusarium odoratissimum (Foc TR4) is a devastating fungal pathogen that causes Panama wilt in Cavendish banana, a high value crop that generates significant revenue. The excessive use of synthetic fungicides for disease control poses risks to both human health and the environment. Consequently, there is a pressing need for eco-friendly alternatives to manage this disease. One potential approach is the utilization of biocontrol agents, which have shown promise in disease control. This study aimed to evaluate the biocontrol potential of fungal endophytes isolated from the naturally-resistant cultivar Cardaba banana against Foc TR4. These fungal isolates were subjected to a dual culture assay, characterized based on morpho-cultural characteristics, and confirmed molecularly using the internal transcribed spacer (ITS) region of rDNA. Results Of the 15 fungal endophytes isolated from Cardaba; Blakeslea trispora, Pseudopestalotiopsis theae, Xylaria badia, Nigrospora hainanensis, Colletotrichum gloeosporioides, Xylaria feejeensis, Gymnoascus reessii, Fusarium subglutinans, Rigidoporus vinctus, and Macrophomina phaseolina showed potential antagonistic activity against F. odoratissimum. Isolates X. feejeensis (USMJMTBL10), G. reesii (USMJMTBL11), and M. phaseolina (USMJMTBR1) had the highest antagonistic activity of 87.8–96.6% against F. odoratissimum in vitro. Conclusion This study presents evidence of the potential of Cardaba-associated endophytes isolated and identified in this research, as effective biocontrol agents against F. odoratissimum, the causal agent of Panama disease in Cavendish banana. Our findings suggest that these endophytes hold promise as a biocontrol agent for managing Foc TR4-infected banana plants. Future research will focus on in planta testing, validation in greenhouse and field trials, as well as development of formulations and application protocols to optimize the utilization of these biocontrol agents in an integrated management approach for Foc TR4-infected bananas.