Background: Biochemistry is a cornerstone of medical education. Its knowledge is integral to the understanding of complex biological processes and how they are applied in several areas in health care. Also, its significance is reflected in the way it informs the practice of medicine, which can guide and help in both diagnosis and treatment. However, the retention of biochemistry knowledge over time remains a dilemma. Long-term retention of such crucial information is extremely important, as it forms the foundation upon which clinical skills are developed and refined. The effectiveness of biochemistry education, and consequently its long-term retention, is influenced by several factors. Educational methods play a critical role; interactional and integrative teaching approaches have been suggested to enhance retention compared with traditional didactic methods. The frequency and context in which biochemistry knowledge is applied in clinical settings can significantly impact its retention. Practical application reinforces theoretical understanding, making the knowledge more accessible in the long term. Prior knowledge (familiarity) of information suggests that it is stored in long-term memory, which makes its retention in the long term easier to recall. Objectives: This investigation was conducted at King Saud bin Abdulaziz University for Health Sciences in Riyadh, Saudi Arabia. The aim of the study is to understand the dynamics of long-term retention of biochemistry among medical students. Specifically, it looks for the association between students’ familiarity with biochemistry content and actual knowledge retention levels. Methods: A cross-sectional correlational survey involving 240 students from King Saud bin Abdulaziz University for Health Sciences was conducted. Participants were recruited via nonprobability convenience sampling. A validated biochemistry assessment tool with 20 questions was used to gauge students’ retention in biomolecules, catalysis, bioenergetics, and metabolism. To assess students’ familiarity with the knowledge content of test questions, each question is accompanied by options that indicate students’ prior knowledge of the content of the question. Statistical analyses tests such as Mann-Whitney U test, Kruskal-Wallis test, and chi-square tests were used. Results: Our findings revealed a significant correlation between students’ familiarity of the content with their knowledge retention in the biomolecules (r=0.491; P<.001), catalysis (r=0.500; P<.001), bioenergetics (r=0.528; P<.001), and metabolism (r=0.564; P<.001) biochemistry knowledge domains. Conclusions: This study highlights the significance of familiarity (prior knowledge) in evaluating the retention of biochemistry knowledge. Although limited in terms of generalizability and inherent biases, the research highlights the crucial significance of student’s familiarity in actual knowledge retention of several biochemistry domains. These results might be used by educators to customize instructional methods in order to improve students’ long-term retention of biochemistry information and boost their clinical performance.