Humulus lupulus L. (HOPs) that is used in the pharmaceutical and industrial area, grow out only in Pazaryeri /Bilecik at Turkey. The aim of this study is to monitor the sustainable health of the HOP agricultural ecosystem through soil and plant microfungal indicators. For this purpose, microfungal biodiversity in soils where HOPs plants are grown and in HOPs plants themselves has been monitored since 2022 in the Pazaryeri/Bilecik region of Türkiye. In this monitoring system, the method followed for soil and plant is as follows: soil samples are taken annually and/or seasonally, while plant samples are taken during the months of March-August, covering the growth and harvest period of HOPs. In determining microfungal biodiversity, Rose Bengal-added Potato Dextrose Agar (PDA-RB), Speziel Nahrstoffarmer agar (SNA), and Selective Fusarium Agar (SFA) + Rose Bengal media were used. The obtained isolates were identified using molecular and morphological methods based on multi-gene region sequencing. In conclusion, Dematiaceous organisms, primarily Aspergillus, Paecilomyces, Penicillium and Taloromyces, Fusarium genera, were recorded in the soil, along with mildew and powdery mildew on plants. This study aimed to reveal the agroecosystem health of HOP by establishing a calendar of this biodiversity over the year and providing findings on how climate variables affect the process. Living Lab applications on this subject are ongoing; steps are being taken to understand the ecological conditions and their consequences through training and working processes with local authorities and farmers.