Fluoroquinolones are broad-spectrum antibiotics that are considered as first line drugs to treat infectious diseases. In order to find out useful fluoroquinolones, the antibiotic resistance of fluoroquinolones, namely, ofloxacin (OFL), ciprofloxacin (CIP), norfloxacin (NRF), enoxacin (ENX), pefloxacin (PFL) and levofloxacin (LVF) was investigated against ninety five clinical isolates that includes Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis. In vitro activity of these isolates was carried out by agar dilution method. All Staphylococcus aureus were sensitive to OFL at 2 μg/ml. About 6% isolates of Klebsiella pneumoniae were found to be resistance to LVF and ENX, 6% to CIP, OFL and PFL and none of the isolates were resistant to LVF and ENX. Percentage resistance of P. aeruginosa was found to be 4.35% to CIP, 7% to OFL and 2.2% to NRF, whereas 8.69% to ENX, 0% to PFL and 17.4% to LVF, respectively. The present study provides the data about the emergence of resistance to fluoroquinolones among gram positive and gram negative bacteria and strongly recommends the rational and appropriate use of these antibiotics.