A new series of 4-((7-methoxyquinolin-4-yl) amino)-N-(substituted) benzenesulfonamide3(a–s)was synthesized via the reaction of 4-chloro-7-methoxyquinoline1with various sulfa drugs. The structural elucidation was verified based on spectroscopic data analysis. All the target compounds were screened for their antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and unicellular fungi. The results revealed that compound3lhas the highest effect on most tested bacterial and unicellular fungal strains. The highest effect of compound3lwas observed againstE. coliandC. albicanswith MIC = 7.812 and 31.125 µg/mL, respectively. Compounds3cand3dshowed broad-spectrum antimicrobial activity, but the activity was lower than that of3l. The antibiofilm activity of compound3lwas measured against different pathogenic microbes isolated from the urinary tract. Compound3lcould achieve biofilm extension at its adhesion strength. After adding 10.0 µg/mL of compound3l, the highest percentage was 94.60% forE. coli,91.74% forP. aeruginosa,and 98.03% forC. neoformans. Moreover, in the protein leakage assay, the quantity of cellular protein discharged fromE. coliwas 180.25 µg/mL after treatment with 1.0 mg/mL of compound3l, which explains the creation of holes in the cell membrane ofE. coliand proves compound3l’s antibacterial and antibiofilm properties. Additionally, in silico ADME prediction analyses of compounds3c,3d,and3lrevealed promising results, indicating the presence of drug-like properties.