The research delves into quinazolinone derivatives, a significant class of heterocyclic compounds. With a resilient quinazolinone nucleus, these derivatives have been explored for their pharmacological potential. Computational docking studies reveal their promise as DNA gyrase inhibitors, with compound (Nitro substituent) displaying a notable -8.3 kcal/mol docking score. Binding energy analyses and receptor interactions highlight their potential . The study's core involves synthesizing Quinazolin-4(3H)-one derivatives with variations at the 6th position. NMR and IR spectral data confirm distinctive structural characteristics. These derivatives exhibit potent antimicrobial activity against gram-positive and gram-negative bacteria, including E. coli, S. typhi, B. subtilis, and S. aureus. Notably, nitro-substituted derivatives demonstrate substantial antibacterial efficacy, indicating a promising avenue for future antimicrobial drug development. Overall, the research underscores the diverse and promising applications of quinazolinone derivatives in pharmacology and therapeutic interventions.