Two carboxamides, N,N'-(1,2-phenylene) bis(quinoline-2-carboxamide) (H2bqb) and N,N'-(1,2-phenylene) dipicolinamide (H2bpb), were synthesized using the ionic liquid tetrabutylammonium bromide (TBAB) as an environmentally benign reaction medium. These compounds were structurally characterized using FT-IR, UV–Vis, and 1H NMR spectroscopy. The interactions of H2bqb and H2bpb with human serum albumin (HSA) were evaluated to assess their structural effects on binding affinity experimentally under physiological conditions through absorbance titration, fluorescence spectroscopy, circular dichroism (CD), and molecular docking (MD) analyses. Fluorescence titration experiments revealed strong complex formation between the carboxamide ligands and HSA, with binding constants of 1.19 × 10¹² M-1 for H2bqb and 9.4 × 10¹¹ M-1 for H2bpb, indicating a static quenching mechanism. Molecular docking analyses showed that both ligands bind to site I (subdomain IIA) of HSA through a combination of intermolecular hydrophobic and electrostatic interactions. CD analysis revealed that these interactions resulted to alterations in the secondary structure of HSA. The higher binding affinity and more favorable interaction profile of H2bqb compared to H2bpb can be attributed to its larger hydrophobic surface area and more favorable spatial arrangement of functional groups, enhancing its interaction with the binding site. Furthermore, the in-vitro anticancer efficacy of H2bqb and H2bpb was evaluated against human colon cancer cells (HT-29) using MTT assays. H2bqb exhibited superior anticancer efficacy after 12, 48, and 72 h of incubation with the HT-29 cell line. Additionally, physicochemical properties and ADMET profiles was analyzed to gain deeper insights into their pharmacokinetic and toxicological characteristics. The results confirmed favorable pharmacokinetic behavior and acceptable toxicity profiles for both ligands, particularly highlighting H2bqb as a promising candidate due to its superior bioavailability, enhanced stability, and potent anticancer activity. These findings suggest that H2bqb is a promising therapeutic agent for colon cancer treatment, owing to its strong binding affinity to HSA and potent cytotoxic effects.