The approaches outline the synthesis of a new series of chalcone using cyano- and chloro-substituted benzaldehydes and 4-fluoro-3-methylacetophenone. The structure of the produced compounds was characterized using elemental analysis and spectral (1H NMR, FT-IR, and UV) methods. In vitro testing was done on the substances that had antibacterial and antifungal activities. All the compounds displayed good binding affinities with the target microorganism proteins, although compounds H and I displayed the strongest affinities, according to molecular docking. The synthesized compounds in vitro antimicrobial activity against Escherichia coli (MCC 2412), Staphylococcus aureus (MCC 2408), Bacillus subtilis (MCC 2010), Pseudomonas aeruginosa (MCC 2080), Saccharomyces cerevisiae (MCC 1033), and Candida albicans (MCC 1439) was examined using the discs diffusion method. The yeast Candida albicans was the target of antifungal action in substances.