The identification of novel anti-infective agents of synthetic and natural origin represents one of the main aims of contemporary drug discovery. In the current work, four different varieties of Nicotiana tabacum, namely, K399, SPG28, Swat No. 1, and Swat No. 2, were studied to assess the antibacterial and antifungal properties of their extracts. The extracts contain anthraquinones, alkaloids, saponins, terpenoids, tannins, resins, steroids, proteins, and carbohydrates, and the antibacterial and antifungal activities were evaluated toward four bacterial and four fungal strains. N. tabacum K399 showed the highest zone of inhibition against E. coli. Similarly, K399 showed the highest antifungal potential, as the highest zone of inhibition for the set was detected against C. albicans. Then, the underlying molecular mechanism was further investigated, and the extracts were tested for their inhibitory potential against urease, an enzyme which is conserved in bacteria and fungi. Additionally, computational tools were enrolled to assess the role of rutin and chlorogenic acid, which are among the main constituents of N. tabacum leaves, in interacting with urease through molecular docking. Combined together, the computational and experimental results support the antibacterial and antifungal potential of N. tabacum extracts, particularly, that obtained from K399 variety.