Background: The association between medication error incidence and time (day shift vs night shift) have not been extensively studied in Saudi Arabia, this study aimed to answer this question: is there a relationship between medication error incidence and time of the day (day shifts vs night shifts) on weekdays and weekends?. Objective: To identify whether medication errors and their sub-categories are significantly different between day shifts, night shifts, during weekdays and weekends. Methods: A retrospective analysis of medication errors reported by health-care practitioners from January 2018 to December 2019 through the Electronic-Occurrence Variance Reporting System (E-OVR) of a university teaching hospital in Riyadh, Saudi Arabia. Statistical analysis was used to determine the differences between the medication errors and their subcategories and day and night shifts during weekdays (from Sunday to Thursday) and weekends (Friday and Saturday). Results: A total of 2626 medication errors were reported over 2 years from January 2018 to December 2019. The most prevalent sub-category of medication errors was prescribing errors (55%), while the least common sub-category of medication errors was administration errors (0.6%). There was a statistically significant difference between medication errors and day of the week. Medication errors that happened on weekdays were greater than at weekends (P = 0.01). During weekends, medication errors were more likely to occur at the night shift compared to the day shift (P < 0.05). Conclusion: Timing of medication errors incidence is an important factor to be considered for improving the medication use process and improving patient safety. Further researches are needed that focus on intervention to reduce these errors, especially during night shifts.