A new series of novel tert-butyl 2,4-disubstituted carboxamido phenylcarbamate derivatives were synthesized and evaluated for their biological activities against three gram positive bacteria viz; Bacillus licheniformis, Bacillus subtilis, Staphylococcus aureus, three gram negative bacteria viz; Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa & four fungi strains viz; Aspergilus niger, Candida albicance, Fusarium oxysporum, Fusarium solani. All compounds were characterized by IR , 1H NMR, 13C NMR, MS and elemental analysis. Among all the title compounds 6b, 6c, 6e, 6g & 6h displayed the most potent antimicrobial activity. The compounds 6b & 6e showed highest antibacterial effect with respect to standard ciprofloxacin. The remaining analogues were showed good to moderate antibacterial activity. The compounds 6b, 6c & 6e showed promising antifungal activity with respect to standard Nystatin. The molecular docking studies were performed to newly synthesized carbamates (6a-k) into the active site of Mycobacterium tuberculosis enoyl reductase (InhA) complexed with 1-cyclohexyl-N-(3,5- dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (2H7M), Candida albicans dihydrofolate reductase complexed with NADPH and 6-methyl-5-[3-methyl-3-(3,4,5-trimethoxyphenyl)but-1-yn-1-yl]pyrimidine-2,4-diamine (UCP115A) (3QLS) to determine the probable binding modes.