Quinazolinone derivatives and hybrids are among the most important classes of N-heterocyclics with broad-spectrum biological activity due to their stability and lipophilicity, which facilitates their passage through the blood-brain barrier and renders them appropriate for addressing various disorders of the central nervous system. Hence, there is a demand for the synthesis of quinazoline based scaffolds suitable for medicinal applications. Pyridine-quinazolin-4(3H)-one hybrids 25a-p have been synthesized, and their antimicrobial and anticancer activities were investigated. Similarly, their binding interactions with Staphylococcus aureus dihydrofolate reductase were computed. Compounds 25a-p were moderately active towards Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginous, and Candida albicans. Compound 25b has the highest inhibition zone of 17 ± 0.5, 15 ± 2 and 19 ± 0.5 mm against MRSA, Pseudomonas aeruginosa, and Candida albicans, respectively. The lowest MIC value (0.625 mg/ml) was observed in 25d. Similarly, a cytotoxic investigation against HL60, MV411, K562, and KG1a leukaemia cell lines revealed compound 25k as the most active with IC₅₀ (4.36 μM) against K562. Compared to the native ligand (7-(2-methoxyphenyl)quinazoline-2,4-diamine) (-8.453 Kcal/mol), molecular docking studies revealed better docking  scores -8.855, -8.865, -8.829, -8.526, -8.980, -8.730, -8.753, -8.623, and -8.622 Kcal/mol in compounds 25a-f, 25h, 25k and 25i, respectively. The most potent, based on the docking score was found to be 25e (-8.980 Kcal/mol), which suggests a stable and strong interaction with the protein.