Emerging resistance to current antibiotics is a global threat to human health. Therefore, comprehending the mechanism behind antibiotic resistance holds paramount importance. In the pursuit of finding new antibacterial agents, our group has developed a small molecule, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole), having bisbenzimidazole as a pharmacophore, targeting bacterial type IA topoisomerase, a novel drug target in bacteria. We examined the emergence of mutations leading to PPEF resistance in laboratory-evolved strains. The growth curve revealed that 25923 PPEF-resistant (SA-PR) and methicillin-resistant 43300 PPEF-resistant (MRSA-PR) attained stationary phase earlier than their respective reference strains. RNA sequencing analysis revealed that (ATP synthase gene) was downregulated by 2 log-fold in both SA-PR and MRSA-PR strains, whereas there was 10 to 13 log-fold downregulation of (methicillin resistance-inducing gene), (bleomycin resistance-inducing gene), (beta-lactamase), (penicillin-binding protein gene), (rRNA adenine methyltransferase gene), and (potassium-transporting ATPase) in the MRSA-PR strain. Quantitative reverse-transcriptase PCR data confirmed these results. Additionally, MRSA-PR showed a 5 log-fold upregulation of and a 9 log-fold downregulation of , indicating increased genomic variability and stress adaptation contributing to resistance. Genomic sequencing revealed deletions of resistance genes, including , , , and in MRSA-PR, resulting in a gain in resistance and a diminishing returns epistasis pattern in PPEF-evolved strains. This led to the development of an evolved MRSA-PR strain susceptible to oxacillin, ciprofloxacin, gentamicin, and imipenem. Our findings indicate that adaptation to PPEF has increased antibiotic susceptibility, thereby changing the clinical outcomes of infections.IMPORTANCEThis study investigates how bacteria, including methicillin-resistant (MRSA) strain, develop resistance to a new candidate antibacterial compound, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole). The research found that resistant strains grew slower and showed significant changes in the activity of genes related to antibiotic resistance. Some resistance genes were deleted in the resistant MRSA strain, making it more sensitive to other antibiotics like oxacillin and ciprofloxacin. These findings highlight how resistance to PPEF leads to increased sensitivity to conventional antibiotics. This suggests that developing combination therapies of PPEF with other antibiotics could optimize treatment regimens and slow resistance evolution. This study also indicates that the antibiotic regimens could be designed to force resistant bacteria into evolutionary trade-offs, where they lose resistance to widely used antibiotics while gaining resistance to a new compound like PPEF.