Different series of thiazole-based derivatives were prepared in this work including thiazolidinone, thiourea, amide, sulfonamide, imidazothiazole, and thiazolopyrimidine derivatives. They were tested for their antimicrobial activity against the G+ve bacteria Staphylococcus aureas, and the G-ve bacteria Escherichia coli and Klebsiella pneumoniae utilizing ampicillin and gentamicin as reference antibacterial drugs. In addition, their antifungal activity was assessed against Candida albicans utilizing fluconazole as a standard antifungal drug. The most active compounds in the antimicrobial evaluation were further subjected to enzyme assay and the results revealed that the thiourea derivative 35, and the 4-nitrobenzamide 38 were the most potent inhibitors to DNA gyrase and topoisomerase IV. They exhibited IC₅₀ values of  25.7 and 30.4 µM respectively against DNA gyrase and topoisomerase IV in comparison with 24.5 and 24.4 µM for ciprofloxacin as a standard drug. Molecular modeling studies were also carried out for the prepared compounds, including docking into the studied enzymes active site. Results explained the superior binding of compounds 35 and 38 with the corresponding enzymes.