A series of chalcones derivatives were synthesized and evaluated for cytotoxic and antibacterial activities in vitro. These modifications changed their bioactivity profile and indicated a combination of SAR analysis toward the substituents in rings A and B of chalcones. Compounds 2, 6–8, 14–17, and 32 exhibited good cytotoxic properties against two human cancer cell lines HT29 and SGC7901. Compounds 16 and 17 showed high antibacterial activity toward 14 clinically isolated multidrugresistant strains. Subsequently, the structure of bi-bioactive compound 16 was determined using single-crystal X-ray diffraction. This study presents a few novel leading compounds for the development of potential antitumor and antibacterial agents.