Mycoplasma contamination remains a major concern in the biopharmaceutical industry especially in tissue culture based viral vaccine and its presence and/or its endotoxin-like metabolites in the final products can result in pyrogenic responses ranging from fever and chills, to irreversible and fatal septic shock. This study was conducted by in vitro screening of mycoplasma in the different ingredients used in production of Foot and mouth Disease (FMD) vaccine using Polymerase Chain Reaction (PCR) using universal primers that are specific to the 16S rRNA region. Tested items include growth media, cell lines, trypsin, seed virus and working virus. Also the study evaluates the inhibitory effect of different concentrations of neomycin, kanamycin, gentamycin, polymyxin B and ciprofloxacin on mycoplasma contaminated cell lines. Our results showed that the prepared growth media, trypsin, seed virus as well as working virus were mycoplasma free and three tested cell lines were also free while another two lines were mycoplasma positive. The mycoplasma positive cell line are poorly grown in comparison with the free line using the same growth media and the virus yield from the apparently normal contaminated line was very low. Ciprofloxacin can be used for treating valuable cell line after 12 days in 25mg/L and after 18 days in 10mg/L. Ciprofloxacin plus regular antibiotic may keep the line sterile for prolonged time but treatment of contaminated cell line is not advisable. So, prophylactic control by strict personal hygiene and personal protective equipment (PPE) and adopt appropriate aseptic techniques is the core solution.