• New isatin Schiff base derivatives have been synthesized as potent antimicrobial agents. • The compounds were docked at the active site of PDF enzyme (PDB ID: 1G2A ) to predict the possible mode of action of compounds. • Docking studies indicated the stable ligand-protein complexes formed by all compounds. • The TOPKAT protocol in DS 3.0 software showed lower toxicity of the designed isatin Schiff base derivatives. • Antimicrobial study and regression analysis have been done for all compounds. A new series of novel Schiff bases has been designed, synthesized and tested for in vitro antimicrobial activity. QSAR studies followed by antibacterial screening using broth dilution technique showed excellent MIC values against four human pathogens, namely Escherichia coli, Pseudomonas aeruginosa, Bacillus cerus and Staphylococcus aureus. A great synergistic effect was observed when the test compounds were used in combination with chloramphenicol and ciprofloxacin. The MIC of the combination was reduced up to 1/4 th or 1/32 nd of the original value, indicating thereby that the combination was 4 to 32 times more potent than the test compounds. Molecular modeling suggested that all ligands interacted well within the active site of peptide deformylase enzyme (PDF) (PDB ID: 1G2A ) and formed stable complexes. Antifungal screening of all compounds was also done using the food poisoning in vitro method on four plant pathogenic fungi, namely Fusarium oxysporum, Aspergillus flavus, Aspergillus niger and Alternaria brassicae. The compounds 8 and 10 showed good activity against F. oxysporum at 100 µg/mL with inhibitory index 70% and 82.5%, respectively and IC 50 value of 2.75 and 2.29 µg/mL, respectively. Better intestinal absorption, oral bioavailability and cell permeation have been predicted for the test compounds using ADMET protocol available within Discovery Studio 3.0 (DS 3.0). The study thus laid the foundation for the development of better antibacterial agents via a combination approach and safe agricultural fungicides.