The rise of bacterial resistance presents a pressing public health challenge, necessitating innovative antimicrobial solutions. Based on our prior work demonstrating the activity of pyrazolo[3,4-d] pyrimidine-based analogues against human dihydrofolate reductase (DHFR), we hypothesized that novel derivatives could function as dual-action antibacterial agents. In this study, we therefore evaluated the capacity of these compounds to both inhibit bacterial DHFR and disrupt quorum sensing (QS)-mediated virulence. The compounds were screened for antibacterial activity against several bacterial strains and the anti-DHF activity was assayed. Anti-virulence potential was assessed in vitro and in vivo, while interactions with the QS targets were investigated using in silico study. Staphylococcus aureus and Pseudomonas aeruginosa were chosen as representative clinically important gram-negative and gram-positive pathogens, respectively, to evaluate the antibacterial and anti-virulence activities. The in vitro and in silico DHFR inhibition assays revealed potent antibacterial activity for the synthesized compounds against various bacteria. Among the most promising candidates, compound 7e exhibited potent antibacterial activity at low minimum inhibitory concentrations (MICs) and demonstrated synergy with conventional antibiotics. The in vitro and in vivo evaluations showed promising anti-virulence activities of the synthesized compounds, particularly 7a and 7e, against P. aeruginosa and S. aureus, respectively. Dynamics simulations showed the strong binding affinity of 7a and 7e to LasI/R and AgrC QS targets in P. aeruginosa and S. aureus, respectively. In conclusion, our findings demonstrate that pyrazolo[3,4-d]pyrimidine analogues, especially 7a and 7e, can function as effective dual-action agents by inhibiting DHFR and suppressing QS-mediated virulence, thus representing a promising new class of anti-virulence therapeutics against priority bacterial pathogens