Kiwifruit Vine Decline Syndrome (KVDS) has become a major concern in Italy, impacting both plant health and production. This study aims to investigate how KVDS affects soil health indicators and the composition of soil microbial communities by comparing symptomatic and asymptomatic areas in two kiwifruit orchards located in Latium, Italy. Soil samples were collected during both spring and autumn to assess seasonal variations in soil physicochemical properties, enzyme activities, and microbial biomass. The results reveal that KVDS influences several soil properties, including pH, electrical conductivity, and the contents of water-soluble carbon and nitrogen. However, these effects varied between orchards and across different seasons. Additionally, KVDS significantly impacts soil enzyme activities and microbial biomass, as assessed through the phospholipid fatty acid (PLFA) analysis, particularly showing an increase in fungal biomass in symptomatic areas. Metabarcoding further demonstrates that microbial communities differ between symptomatic and asymptomatic soils, exhibiting notable shifts in both diversity and relative abundance. Our findings emphasise the complex interactions between plants, soil, and microbial communities in relation to KVDS. This suggests that the syndrome is multifactorial and likely linked to an imbalance in soil microbial communities at the rhizosphere level, which can negatively affect soil health.