Levofloxacin is a member of the fluoroquinolone class of antibacterial agents with a safe and effective treatment. The aim of this study is to evaluate the susceptibility and resistance pattern of clinical isolates causing different types of infections to Levofloxacin. In vitro antibacterial activity and resistance patterns of levofloxacin antibiotic were studied using disk diffusion method. A total 485 strains isolated from various clinical specimens from September 2020 to November 2020 in Al Saleem Medical Laboratory. This is for isolation and identification of pathogenic gram negative and gram positive bacteria. The samples were involving urine, semen, swabs, High vaginal swab, Blood, Stool, CSF, Body Fluid and Sputum. The isolates were identified by biochemical tests as well as the sensitivity and resistant were tested by levofloxacin antibiotic. In present study, gram negative bacteria were prevalent, 75.6% than gram positive bacteria 47.7%. In the four hundred eighty five clinical isolates comprising of Escherichia coli, Klebseilla spp, Staph Aureus, Enterobacter spp, Streptococcus pneumonia, Pseudmonas spp, Streptococcus pyogen, Citrobacter spp, proteus spp and Staph epidermidis were collected from different clinical specimens. The predominantly isolated bacteria were Escherichia coli, followed by Klebseilla spp, staph Aureus and Enterobacter spp. The levofloxacin antibiotic has sensitive against most of the isolates, E Coli (31%), Klebseilla spp (14%), Staph Aureus (8.7), Enterobacter spp (6%), strep pneumonia (5%), Pseudomonas spp (3%), Strep pyogen (1%) and Citrobacter spp (0.6%). Levofloxacin exhibited statistically significantly high bactericidal activity against all strains of gram positive and gram negative bacteria. Levofloxacin was shown to be active against both Gram-positive and Gram-negative bacteria.