Soil enhancements such as biochar (BC) are gaining attention as tools to mitigate climate change and also to promote crop growth. However, biochar use can disrupt soil ecosystems by changing the soil’s physical, chemical, and biological properties. The study aimed to determine how biochar influences soil physical changes such as specific surface area (SSA) and water vapor sorption, and how these conditions affect arbuscular mycorrhizal fungal (AMF) hyphae growth and glomalin production. The study analyzed these factors at different plant phenological phases (i.e., flowering, development of fruit, and ripening of fruit and seed) to better understand the changes within the system while varying biochar amounts. The study also investigated the effect of different soil physical and chemical parameters on mycorrhizal hyphae growth and glomalin production. Four treatments were investigated: 0, 0.5%, 2.5%, and 5.0% (w/w) biochar amended silt loam soil planted with pepper. Soil samples were taken at the beginning and weeks 6, 10, and 12 of the study. The amount of adsorbed water vapor increased with an increasing amount of biochar added to the soils. Compared to control, SSA was significantly higher in all biochar amended treatments based on adsorption data, and only in the highest biochar amended soils for the desorption data at the end of the experiment. The presence of AMF in the roots appeared at week 6 of the experiment and the intensity of AMF root colonization increased with the age of plants. The AMF colonization parameters were significantly lower in BC2.5 compared to all other biochar amended soils. The abundance of intraradical AMF structures was highly correlated with several physicochemical soil parameters, such as SSA, the geometric mean diameter of soil aggregate, soil aggregate sizes, or pH. Glomalin production was negatively correlated with SSA, water vapor adsorption, aggregate stability, aggregate size, total nitrogen, potassium, and organic carbon content of the soil, while positive correlation was observed with bulk density. Increased biochar amount resulted in a significant decrease in glomalin production, concurrent with the age of the plants. Our results highlight the great complexity of interactions between soil physicochemical and biological parameters, and the importance of the time of sampling when biochar is used in soil, as the effects of biochar additions on the plant, soil physical characteristics, and soil microsymbionts vary over time.