Background: Continuous evaluation of students and employee’s knowledge and attitude in clinical laboratories is mandatory to ensure a high level of competency, proper practice and to assess the need for training, which shall be reflected on the quality of laboratory results. The aim of the present study was to evaluate the knowledge, attitude, and practice in microbiology laboratories among employees (at King Fahd Hospital of the University) and clinical laboratory students (at Imam Abdulrahman Bin Faisal University) Methods: This was a cross-sectional survey of 30 2nd year students, 26 3rd year students, 24 4th year students in the Clinical Laboratory Sciences department, and 30 employees. Participants completed a survey comprising 30 questions to assess their knowledge and attitude towards the use of equipment and practice in the microbiology laboratory. Results: The results indicated that there was no significant difference between the average scores of all levels of students regarding their knowledge (p = 0.85, 0.999, and 0.869), attitude (p = 0.883, 0.996, 0.853), and practice (p=0.633, 0.325, 0.858) in the microbiology laboratory. Employees scores (knowledge;5.03±2.646, attitude; 12.03±4.89, and practice; 7.7±6.11) were quite poor, as indicated by the lower average results than that of students (knowledge; 5.65±3.08, attitude; 13.25±5.33, and practice; 13.46±5.7). Conclusions: It is concluded that the knowledge, attitude, and practice of students and employees in the microbiology laboratory needs to be meticulously monitored and improved to ensure high achievement of learning outcomes and better overall performance in the laboratory. This may be achieved through using frequent quizzes and continuous education programs.