Background: The antimicrobial resistance due to biofilm formation among bacteria is a significant problem in the healthcare and food industries. Objective: The current study describes the synthesis of enrofloxacin derivatives 2-17, and the evaluation of their anti-bacterial and anti-biofilm activities. Methods: Compounds 2-17 were synthesized through the acylation of enrofloxacin with thionyl chloride, followed by reaction with different aromatic amines. The new analogues identified among the sixteen compounds were 2-7, 11, 14, and 17. Results: Compound 2 appeared to be effective against pathogens S. aureus as well as K. pneumonia, whereas, compound 11 was found active against K. pneumonia only. Compound 2 inhibited >75% biofilm formation of S. aureus at 20 μg/mL and K. pneumonia at 10 μg/mL concentrations. These doses are far below the bactericidal concentration of compound 2, suggesting the anti-virulence mechanism of these compounds. Compound 11 inhibited 60% biofilm formation of K. pneumoniae at 70 μg/mL concentration. Compound 5 inhibited the biofilm of K. pneumoniae at 62 μg/mL concentration but also had bactericidal properties at this concentration. Interestingly, compound 2 eradicated the preformed biofilm of both the pathogens at much lower doses as compared to control drug, gentamycin and substrate, enrofloxacin. Cytotoxicity of compounds 2–17 was checked by a standard method using 3T3 normal cell lines (mouse fibroblast), all compounds were found to be noncytotoxic. Conclusion: These compounds can be used alone or with FDA approved drugs to overcome biofilm related K. pneumoniae and S. aureus infections.