2-(2-Chloroquinolin-3-yl)-3-(substituted phenyl/pyridinyl)quinazolin-4(3H)-one derivatives 5a-m were synthesized in good yield using conventional as well as microwave heating conditions in presence of L-proline as a catalyst. Synthetic conditions were optimized using an orthogonal array system of Taguchi's design. The structure of synthesized quinazolinone compounds 5a-m was established with the help of physico-chemical analysis and various spectroscopic techniques like FT-IR, mass, 1 H-NMR and 13 C-NMR. Further, all the synthesized compounds were screened for their antimicrobial (antibacterial, antifungal and antimalarial) activities. The results of antimicrobial screening showed that compounds 4g, 4j, 4m, 5e and 5g have good activity against bacterial species, whereas compounds 4d, 4g, 4m, 5b, 5d and 5g have good activity against malaria pathogen. In addition to this, compounds 4i, 4j and 4m exhibited good to moderate activity against fungal strains.