Aims: This study aimed to evaluate the biological potential of Cannabis sativa L. leaf co-products obtained through different extraction methods (sonication, maceration, and decoction), with a particular focus on their antimicrobial properties and chemical composition. Methodology and Results: Extracts were prepared using sonication, maceration, and decoction, and their antimicrobial activity was assessed against two pathogenic microorganisms: Aggregatibacter actinomycetemcomitans and Candida albicans. The sonication extract exhibited the highest antibacterial activity against A. actinomycetemcomitans, whereas the maceration extract showed strong antifungal activity against C. albicans. All extracts, except the decoction, demonstrated both bactericidal and fungicidal effects. Chemical characterization was performed using High-Performance Liquid Chromatography (HPLC), revealing the presence of cannabinoids (notably cannabidiol and HU-331) along with flavonoids, phenols, alkaloids, and dihydrostilbenoids. Additionally, molecular docking analysis predicted strong interactions between several identified compounds and bacterial target proteins, indicating promising drug-like properties. Conclusion, Significance and Impact: These findings suggest that Cannabis sativa L. leaf co-products represent a valuable and underutilized source of bioactive compounds with significant antimicrobial potential. This study highlights their relevance for future pharmaceutical applications and supports their valorization within a sustainable biorefinery approach.