This study aimed to determine factors influencing happiness in preclinical students especially in aspects of teaching and learning, students’ motivation, study habits, and academic achievement. This study was a questionnaire-based cross-sectional study. The questionnaire was validated and approved for clarity, readability, rational analysis, and comprehensiveness by the experts. Questionnaires were sent to all second and/or third-year medical students of the 2018 (328) and 2019 (329) classes, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand. Cronbach’s alpha values were 0.886. The data were analyzed by the Statistical Package for Social Science version 18. From open-ended questions, the top 5 ranking factors increasing students’ happiness were teaching of instructors, content satisfaction, content understanding, free time, and handouts. Factors that had positive influences/associations with happiness of preclinical students were happiness in learning subjects taught in a particular year; satisfaction of content, study outcomes, materials, handouts, teaching motivating desire to learn, and applicability of content to medical profession; high motivation to study medicine, expected score, and the percentage of achievement of study targets; less amount of time spent on recorded-e-lecture study and internet for non-academic use; and low stress. Happiness of preclinical students was influenced by satisfaction in teaching and learning, their attitude, study habits, and academic achievement. Further studies on determining students’ happiness after improvement of teaching and learning environments, augmenting activities that enhance students’ motivation, and promoting good study habits among medical students, are required to prove whether enhancement of these factors could effectively increase students' happiness.