The primary objective of this investigation was to assess the potential applicability of hemp (Cannabis sativa L. var. sativa) lateral inflorescence extract in mitigating the growth of fungi, including phytopathogens, on agricultural plants. The extract, comprising a blend of biologically active compounds, holds promise for integration into contemporary plant protection methodologies. The research involved a comprehensive analysis of the extract’s chemical composition, encompassing the determination of total polyphenol and flavonoid content (utilizing spectrophotometric methods), antioxidant activity (evaluated through the DPPH method employing synthetic 2,2-diphenyl-1-picrylhydrazyl radical), and cannabinoid content (analyzed using HPLC techniques). Additionally, this study employed the poisoned substrate method to gauge the impact of 5, 10, and 20% extract concentrations on the growth of various microfungi, including Alternaria alternata, Botrytis cinerea, Colletotrichum coccodes, Fusarium avenaceum, F. culmorum, F. graminearum, F. oxysporum, F. sporotrichioides, and Trichoderma koningii. The hemp extract demonstrated a substantial presence of total polyphenolic compounds, with polyphenol and flavonoid concentrations measuring 149.65 mg/mL and 1.55 mg/mL, respectively. Furthermore, the extract contained cannabinoids at a concentration of 0.12%. The most pronounced antifungal activity was observed with the 20% extract, particularly against T. koningii (62.22–84.79%), C. coccodes (61.65–81.82%), and B. cinerea (45.00–75.42%). However, the efficacy of hemp extracts exhibited large differences against Fusarium spp. (3.10–72.95%), dependent on the specific extract and fungus strain. Introduction of hemp extracts to the substrate induced a reduction in substrate pigment and a discernible color alteration in the mycelium to a lighter shade compared to the control. These findings mark the initial phase in the exploration of practical applications for plant extracts, setting the groundwork for subsequent field trials to ascertain the extract’s impact on phytotoxicity and the health status of agricultural plants.