Daily routines may influence eating patterns; however, differences in intake on weekdays and at weekends have rarely been explored. Furthermore, these differences have not been analyzed among university students (a particularly interesting group among the younger generations). The aim of the study was to evaluate weekend−weekday variation in the Mediterranean diet among Spanish university students, while investigating the potential influence of age, gender, studies, body mass index, smoking status and physical activity status. A repeated-measurement 28-day cross-sectional observational study with self-reported dietary intake collected using the e12HR app was conducted. There were 361 participants: average age 20.6 years; 72.9% women; 58.2% students of Pharmacy; average BMI 21.9 kg/m2; 91.4% nonsmokers; 77.6% performed ≥150 min/week of physical activity. Outcome measurements were adherence to the Mediterranean diet (AMD) index and percentage of participants meeting recommendations for each food group on weekdays and at weekends. In all subgroups, Spanish university students’ global diet was associated with low AMD, with poorer diet quality (>12% reductions in mean scores of AMD index and >26% reductions in adequate adherence scores (≥9)) at weekends. In conclusion, weekend health behaviors of Spanish university students displayed less favorable eating behavior, making the weekend an important target for public health interventions aiming to improve dietary intake.