Facile synthesis of molecular hybrids containing a 2,4-dinitrophenyl moiety was achieved via nucleophilic aromatic substitution of the fluoride anion of Sanger’s reagent (2,4-dinitrofluorobenzene) with various N, S, and O nucleophiles, considered as bioactive moieties. Antimicrobial evaluation of the new hybrids was carried out using amoxicillin and nystatin as antibacterial and antifungal reference standards, respectively. MIC test results identified the compounds 3, 4, and 7 as the most active hybrids against standard strains and multidrug-resistant strains (MDR) of Staphylococcus aureus , Escherichia coli, and Pseudomonas aurginosa. Most of the hybrids displayed two times the antibacterial activity of AMOX against MDR Pseudomonas aeruginosa, E. coli, and a standard strain of P. aeruginosa (ATCC 29853), while demonstrating a weak antifungal profile against Candida albicans. Selectivity profiles of the promising compounds 3, 4, 6, 7, 8 , and 11 on WI-38 human cells were characterized, which indicated that compound 3 is the safest one (CC 50 343.72 μM). The preferential anti-Gram-negative activity of our compounds led us to do docking studies on DNA gyrase B. Docking revealed that the potential antimicrobial compounds fit well into the active site of DNA gyrase B. Furthermore, in silico absorption, distribution, metabolism, and excretion (ADME) predictions revealed that most of the new compounds have high gastrointestinal absorption and a good oral bioavailability with no BBB permeability.