Quinoline derivatives are attracting considerable interest due to their biological importance. In this paper, several 2-amino-4-aryl-6-(quinolin-2-ylthio)pyridine-3,5-dicarbonitrile derivatives are synthesized by adopting a one-pot reaction of quinoline-2-thione, aromatic aldehydes, and malononitrile in the presence of sodium hydroxide in absolute ethanol. The structures of these newly synthesized compounds were determined using different spectroscopic techniques, including elemental analyses, IR, 1 H NMR, and MS. The synthesized derivatives were screened for their antimicrobial and cytotoxic activities. Compounds 4a, 4b, 4d, and 4e exhibited promising antimicrobial activity compared to antibacterial and antifungal standard drugs. Additionally, 4f, 4d, and 4g showed potent cytotoxic activity against both MCF-7 and A549 cells with IC50 values (6.39-9.3 μM). Our molecular docking results of compound 4f prove good binding affinity toward the three tested proteins as Jak2/STATA3 inhibition and are in accordance with the RT-PCR mRNA expressions of the compound against MCF-7 cells which downregulated the Jak2 and STAT3 genes, and this may be the proposed mode of action for anti-breast cancer activity.