One of the main problems in cell culture unit is mycoplasma contamination. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production, cell therapy and its importance for diagnostic routine work in selected labs, so more concerns about mycoplasma contamination will arise. In our study, a total of 50 cell cultures from Animal Health Research Institute, Dokki, Giza., VACSERA and Veterinary Serum & Vaccine institute Abbasia were monitored for mycoplasma using culture and PCR methodology. The contamination was detected in the cell culture collected from all laboratories. Mycoplasmas were detected by culture in 29/50 (58%) of the cell culture samples which subsequently identified with PCR using Mycoplasma group specific primer detecting all species of mycoplasma which gave bands at 280 bp in all positive culture samples, the most frequent species was M. arginini (30%), followed by M. orale (28%). MDBK were positive only for M. arginini which gave band at 326 bp, while VERO and BHK cells were infected only with M. orale which gave bands at 87bp. Awareness about the sources of mycoplasma and pursuing aseptic techniques in cell culture unit along with reliable detection methods of mycoplasma contamination can provide an appropriate situation to prevent mycoplasma contamination in cell culture.