Our work starts by evaluating the antifungal efficacy of Trichoderma strains (TR04 Trichoderma afroharzianum and TR05 Trichoderma gamsii) and chitosan against Penicillium species, alongside an analysis of fungal contamination in walnut kernels from the Kurdistan Region of Iraq and Hungary. Through a dual culture assay, well diffusion assay, and fungal population study, the experiments provide compelling evidence of the biocontrol potential of these agents and highlight the global challenge posed by fungal pathogens in walnut production. Dual culture assay demonstrated that Trichoderma strains TR04 and TR05 significantly curtailed Penicillium growth on Potato Dextrose Agar (PDA) plates, likely through mycoparasitism and antibiosis, as evidence by reduced Penicillium colony sizes compared to control. The well diffusion assay confirmed chitosan’s effectiveness, with 0.5% and 1% (w/v) solutions producing clear zones of inhibition, attributed to disruption of fungal cell integrity or spore germination suppression. Fungal population analysis revealed that Penicillium and Aspergillus species dominated the microbial profiles of both Kurdistan and Hungarian walnuts, even in visually healthy kernels, indicating a widespread contamination issue with potential mycotoxin risks. These findings validate Trichoderma and chitosan as sustainable biocontrol tools for managing Penicillium in walnuts, offering eco-friendly alternatives of chemical treatment. The prevalence of Penicillium and Aspergillus across regions underscores the need for proactive, globally coordinated strategies to ensure walnut safety and quality. Recommendations include integrating Trichoderma and chitosan into walnut production, enhancing fungal monitoring, pursuing further research to optimize biocontrol applications, and fostering international collaboration to address fungal challenges effectively. The walnut samples offer divers opportunities for food applications, with TEKI and KOKI excelling in dry matter and TPC, TEKO leading in flavonoids, and KOKO providing consistency. PEKO and PEKI, while less distinguished, remain viable for general uses, though PEKI requires processing improvements. By aligning product development, quality control, and labeling with these profiles, stakeholders can optimize nutritional and commercial outcomes. TEKI and KOKI, with high dry matter and TPC, are prime candidates for antioxidant rich, premium products, while TEKO’s flavonoid richness suits functional foods. KOKO’s uniformity supports low fat applications, and PEKO is best for general uses. PEKI’s variability and lower content require processing enhancements. Recommendations include stringent quality control for TEKO, clear nutritional labeling to emphasize KOKI and TEKO’s antioxidant benefits, and further research to address variability and explore functional properties, ensuring these samples meet diverse market needs. The elemental analysis demonstrates that PEKI and PEKO exhibited the highest levels of key elements, indicating rich profile. TEKI excelled in copper and magnesium, while KOKO led in iron and showed consistent calcium and zinc content. KOKI had lower magnesium and calcium and TEKO recorded the lowest potassium with higher variability in phosphorus and calcium. Dry matter adjusted values highlighted PEKI’s nutrient density and KOKI’s lower magnesium. The absence of detectable toxic elements confirms the samples’ safety, supporting their use in nutrient dense food products, with PEKI and PEKO ideal for health focused applications and KOKO suited for consistent formulations.