In this study, novel series of chalcone derivatives, namely, 4-[4-(3-phenyl-acryloyl)-phenylamino]-chromen-2 one (5a–k) have been synthesized from the intermediate 4-(4 acetyl-phenylamino)-chromen-2-one (4). Cyclization reaction of chalcone (5a–k) with hydrazine hydrate, guanidine nitrate, and malononitrile gives the corresponding 4-[4-(1 acetyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-phenylamino]-chromen-2-one (6a–k), 4-[4-(2-amino-6-phenyl-pyrimidin-4-yl)-phenylamino]-chromen-2-one (7a–k), and 2-amino-6-[4-(2-oxo-2H-chromen-4-ylamino)-phenyl]-4 phenyl-nicotinonitrile (8a–k) derivatives were synthesized. The newly synthesized compounds were evaluated for their antimycobacterial activity and antimicrobial activity against eight bacteria (S. aureus, B. cereus, E. coli, P. aeruginosa, K. pneumoniae, S. typhi, P. vulgaris, and S. flexneri) and four fungi (A. niger, C. albicans, A. fumigatus, and A. clavatus).