Background Although many studies have recently been published on the value of serious games for medical education, little attention has been given to the role of dark play (choosing unacceptable actions in games). Objective This study aimed to investigate potential differences in the characteristics of medical students who have the opportunity to choose normal or dark play in a serious game. This study also aimed to compare their reasons for choosing a play strategy and their perceptions of what they learned from their game play. Methods We asked undergraduate medical students to play a serious game in which they had to take care of a patient with delirium (The Delirium Experience). After getting acquainted with the game, students could opt for normal or dark play. Student characteristics (age, gender, experience with caring for older or delirious patients, and number of completed clerkships) were collected, and the Delirium Attitude Scale and Learning Motivation and Engagement Questionnaire were administered. Reasons for choosing normal or dark play were evaluated with an open-ended question. Information on lessons they had learned from the game was collected using an open-ended question and self-reported knowledge on delirium. Results This study had 160 participants (89 normal play, 71 dark play). Male students (26/160, 56.5%) chose dark play significantly more often than female students (45/160, 39.5%; P=.049). We did not find significant differences in student characteristics or measurement outcomes between play strategies. Participants’ main reason for choosing normal play was to learn how to provide care to delirious patients, and the main reason for dark play was to gain insight into what a delirious patient has to endure during delirious episodes. All participants learned what to do when taking care of a delirious patient and gained insight into how a patient experiences delirium. We found no differences in self-reported knowledge. Conclusions When medical students have the opportunity to choose dark play in a serious game, half of them will probably choose this play strategy. Male students will more likely opt for dark play than female students. Choice of play strategy is not affected by any other student characteristic or measurement outcome. All students learned the same lessons from playing the game, irrespective of their learning strategy.