This study investigates the impact of chemical and biological treatments on the mycobiota of winter wheat seeds and the fungal composition of the soil. Conducted in 2023-2024 using the winter wheat variety Aliot, the research aimed to determine how different seed treatments affect the microbial populations and the development of wheat seedlings. The experiment was carried out in Sumy, Ukraine, at the Educational Research and Production complex of Sumy National Agrarian University. Chemical treatments tested included Tebuzan Ultra F.C.S., Celest Top 312.5 FS TH, Maxim 025 FS TH, and Record F.C.S., while biological agents included Azotobacterin-K BI, ECOSTERN Trichoderma CS, Bacillus megaterium, and others. Seeds were treated and their mycobiota were analyzed using biological methods to assess changes in fungal and bacterial populations. Additionally, treated seeds were sown to study the effects on the soil microbiota. The results showed that chemical treatments effectively suppressed several fungal species, particularly Alternaria tenuissima, and led to an increase in bacterial colonies within the seeds. However, they also reduced microbial diversity in the soil, which in some cases negatively impacted seedling development. In contrast, biological treatments, while less effective at completely suppressing fungal pathogens, enriched the seed microbiota, particularly increasing Aspergillus oryzae populations, and promoted the growth of beneficial soil microorganisms. Biological agents such as Azotobacterin-K BI and ECOSTERN Trichoderma CS significantly improved seedling length, reaching up to 13.59 cm, compared to chemical treatments which sometimes reduced seedling growth. Overall, the study highlights that while chemical treatments provide immediate protection against pathogens, biological treatments offer long-term benefits by enhancing microbial diversity and promoting healthier plant growth. These findings support the growing trend toward sustainable agriculture, where biological agents can be integrated to reduce chemical inputs and improve environmental safety without compromising yield.