The laboratory personnel in hospitals are at risk in terms of transmission of various infectious diseases. The aim of this study is to evaluate the knowledge, behavior and attitude of the health personnel who work in one university and two state hospitals in Izmir, Turkey, about biosafety. The study is an observational-sectional study. Participants were selected via random sampling method. The hospitals were visited on workdays determined by the random selection method and all of the personnel (doctor, technician, cleaning-staff) were included to the study. The data were analyzed statistically using Chi square test. Of the 183 participants included in the study, 106 were from Dokuz Eylül University School of Medicine Central Laboratory and 77 were from state hospitals. 62.8% of the participants were female, 37.2% were male and mean age of all was 32.8 +/- 6.9 years. 23.5% of the participants stated that they had previously taken education about biosafety (p= 0.002). It was determined that 91.3% of the participants were wearing gloves and 87.4% of them were wearing lab-coat during laboratory studies. A significant difference was observed between the hospitals in terms of use of gloves (p= 0.004). All the participants stated that they wash their hands and 43% of them indicated that their daily hand wash rate was > or = 10 times. It was determined that 38.3% of the participants consumed food or drinks in the laboratory, however, this rate was statistically significantly less in the university hospital laboratory (p= 0.000). The rate of participants who had been subjected to a microorganism in the last six months was 6.6%. Obedience to the biosafety rules in laboratory will not only provide a safer environment but also improve the quality of work. We believe that the results of this study will serve as a guide for future studies on laboratory biosafety.