Introduction: Workers in clinical laboratories are exposed to occupational hazards on a daily basis and their health and safety may be threatened if appropriate protective standards are not implemented. The aim of this study was to assess the knowledge and practices of clinical laboratory workers towards biosafety measures, in Al-Madinah city, Saudi Arabia. Methodology: Clinical laboratory staff was recruited from both the public and private sectors. A structured self-administered questionnaire was used to achieve the aim of the study. Results: A total of 208 workers participated in the study (64% were males, 57% were from the public sector and 71% held a BSc degree). About 68% of the workers were trained in laboratory safety. The majority (> 80%) followed guidelines for disposing medical wastes, decontamination of sample spills, and use of protective lab coats, gloves, etc. However, among participants, 24.2% used to eat, drink or use gum, 18.3% used cosmetics and 24.6% used the mobile phone in the lab. About 18.4% reported that they continued working with a finger cut, whereas 67% reported that they used to recap needles after blood withdrawal. These unacceptable behaviors were associated with lack of lab safety training (P < 0.05), biology degree holders (P < 0.05), and low experience (3 years and less, P < 0.01). With respect to facilities, most of the laboratories complied with standard safety measures. Conclusion: The majority of the sample showed good laboratory practices with respect to safety measures. However, some behaviors are not accepted and need interventions