The aim of this study was preparation of new derivatives based on 2-((4-chlorophenoxy)methyl)-N-(arylcarbamothioyl)benzamide structure; the new compounds were characterized by IR, NMR (1H, 13C) spectroscopy, and elemental analysis. The obtained compounds were evaluated for their in vitro antimicrobial activity against planktonic and biofilm-embedded microbial cells (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans), by qualitative and quantitative assays. Some of the compounds revealed promising antibacterial and antifungal activities, with low minimum inhibitory concentration values between 0.15 and 2.5 mg/mL and minimal biofilm eradication concentrations of 0.019–2.5 mg/mL. To investigate the potential target of their antibacterial activity, in silico drug-likeness and molecular docking screenings on Staphylococcus aureus DNAgyrase were performed. The compound with the best antibacterial activity (1g) was docked into topoisomerase II DNA gyrase enzymes (PDB ID: 2XCS) and showed valuable interactions with the target protein along with good docking scores, suggesting that it can act by the inhibition of DNA replication. The tested compounds exhibited only a poor antioxidant activity, as revealed by the in vitro assay using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.