The ideal synthesis techniques and the biological activity analysis of 2-(4-nitrophenyl)quinazolin-4-one are presented in this work. Electrophilic substitution processes were used to create the chemical, and several catalysts and solvents were used to optimize the reaction conditions. Using spectroscopic methods including Raman spectroscopy, the structure of the produced molecule was verified. The anthelmintic, antibacterial, antifungal, and cytotoxic qualities of 2-(4-nitrophenyl)quinazolin-4-one were assessed in terms of its biological activity. The findings show that this substance has a high level of biological activity, especially when it comes to its antibacterial and antiparasitic properties. In order to establish relationships between the molecular structure and biological activity and to provide information for future drug development and quinazolin-4-one derivative optimization, structure-activity relationship (SAR) investigations were also carried out.