Background: Continuous evaluation of students and employee’s knowledge and attitude in the clinical laboratory is mandatory to ensure a high level of competency, proper practice and to assess the need for training. This shall be reflected on the quality of laboratory results. Objective: the aim of the present study was to evaluate the knowledge, attitude, and practice of employees and students who belong to the department of Clinical Laboratory Sciences in the standard microbiology laboratory towards the use of laboratory equipment. Methods: This is a cross-sectional survey in which thirty 2nd year students, twenty-six 3rd year students, twenty-four 4th year students in the Clinical Laboratory Sciences department and thirty employees completed a survey comprising 30 questions to assess their knowledge and attitude towards the use of equipment and practice in the microbiology laboratory in order to assess the achievement of past learning outcomes. Results: The results indicated that there was no significant difference between the average scores of all levels students regarding their knowledge, attitude, and practice in the microbiology laboratory. Employees scores were quite poor, as indicated by the lower average results than that of students. Conclusion: It is concluded that the knowledge, attitude, and practice of students and employees in the microbiology laboratory need to be meticulously monitored and improved using frequent quizzes and continuous education programs to ensure high achievement of learning outcomes and better overall performance in the laboratory.