The purpose of this study was to assess the health-related quality of life (HRQoL) in a sample of Croatian university students and to determine its association with sociodemographic and lifestyle characteristics. The study was conducted on a random sample of 1750 students in Zagreb, Croatia. We used the 12-item Short-Form Health Survey (SF-12v2) and separate questions on sex, age, body mass index (BMI), settlement size, disposable income, cigarette consumption, alcohol consumption, and exercise frequency. The HRQoL scale on which Croatian students scored the highest was Physical Functioning. High mean scores were also found for other physical health measures, while the scores in mental health scales were somewhat lower. Male students scored significantly higher than females on all SF-12 scales (p < 0.05). The regression analysis showed that Physical Component Summary score (PCS) is negatively related to BMI (partial r = −0.09) and cigarette consumption (partial r = −0.06), and positively related to alcohol consumption and exercise frequency (partial r = 0.08, for both), while Mental Component Summary score (MSC) is positively related to BMI (partial r = 0.07) and exercise frequency (partial r = 0.10). Although HRQoL-scores for the Croatian students were higher than in the general population of Croatia and student populations in several other countries, there is still room for improvement, especially in the mental health aspect. In general, HRQoL was found to be significantly lower among females and smokers, but positively related with exercise frequency. In order to improve students’ HRQoL, university authorities should consider initiating health-promotion programs with emphasis on females, smokers and non-exercisers.