Apple bull’s eye rot (BER), caused by Neofabraea representatives, is a major challenge for apple production. This study aimed to identify microbial antagonists capable of suppressing Neofabraea by leveraging soil microorganisms from six distinct apple orchard land management systems. Key candidates, Bacillus velezensis (B134/22, B233/22, B267/22) and Trichoderma koningiopsis (G779/22) inhibited 65% of Neofabraea isolates, indicating their strong potential for biocontrol. T. koningiopsis produced chitinase and β-glucanase, while B. velezensis relied on glycogen degradation, suggesting diverse biocontrol strategies. Antibiotic resistance profiles showed minimal resistance genes, reducing environmental risks. Sensitivity to fungicides was assessed, with T. koningiopsis sensitive to Siarkol and B. velezensis to Zato and Luna fungicides, supporting their compatibility with orchard practices. Tested isolates showed susceptibility to some chemical substances used in apple orchard practice (e.g. antibiotics, copper(II) sulfate, fusidic acid, or promethazine). These findings highlight the importance of integrating biocontrol agents with agrochemical use, ensuring sustainability in apple production while minimizing environmental impact.