ESKAPE pathogens are notorious in causing nosocomial infections and escaping current antibiotic treatments. There has been a dramatic increase in nosocomial infections accompanied with a decrease in the number of antibiotics developed, leading to significant increase in morbidity and mortality among patients. In an attempt to combat this problem, derivatives of ciprofloxacin, rifabutin and beta-lactam antibiotics were synthesized and tested against the ESKAPE pathogens. From minimum inhibitory concentration assays, 4 ciprofloxacin analogs and 8 beta-lactam analogs were found to be effective against multiple bacterial species. Additionally, 12 rifabutin analogs and 23 beta-lactam analogs were potent against single bacterial species, primarily toward methicillin-resistant Staphylococcus aureus (MRSA) at a concentration of ≤ 25 µg mL-1. Based on the effectiveness against methicillin-resistant Staphylococcus aureus (MRSA), three rifabutin analogs were selected for further testing. Two rifabutin analogs (DU644 and DU645) were found to possess between a one to twofold mean increase of inhibitory activities, while the other rifabutin analogs (DU650) demonstrated up to a twofold decrease of inhibitory activity when compared to the parent drug. These compounds were then examined for their bactericidal and antibiofilm activities against MRSA. From these assays, we found that DU644 and DU645 were 4 times more bactericidal and antibiofilm against MRSA when compared to the parent drug. In addition, rpoB mutation validation results confirmed that modification of these rifabutin derivatives at the C3 and C4 positions, and bearing an imidazolyl ring carrying substituted spiropiperidyl ring, did not change their mechanism of action towards the beta-subunit of RNA polymerase. Cytotoxicity testing performed using human hepatocellular carcinoma epithelial cells (hepG2) showed that at concentrations ranged from 1.25 µg mL-1 to 25 µg mL-1, DU644 and DU645 showed very low toxicity. Collectively, structural drugs modifications of these obsolete drugs are able to restore their antibacterial activities against MRSA, which is notable as the most infectious nosocomial pathogen. Therefore, further development and application of rifabutin analogs might be beneficial for medical use to combat MRSA infections.