Background: The independent and combined influence of smoking, alcohol consumption, physical activity, diet, sitting time, and sleep duration and quality on health status is not routinely examined. This study investigates the relationships between these lifestyle behaviors, independently and in combination, and health-related quality of life (HRQOL). Methods: Adult members of the 10,000 Steps project (n = 159,699) were invited to participate in an online survey in November-December 2011. Participant socio-demographics, lifestyle behaviors, and HRQOL (poor self-rated health; frequent unhealthy days) were assessed by self-report. The combined influence of poor lifestyle behaviors were examined,independently and also as part of two lifestyle behavior indices, one excluding sleep quality (Index 1) and one including sleep quality (Index 2). Adjusted Cox proportional hazard models were used to examine relationships between lifestyle behaviors and HRQOL. Results: A total of 10,478 participants provided complete data for the current study. For Index 1, the Prevalence Ratio (p value) of poor self-rated health was 1.54 (p = 0.001), 2.07 (p#0.001), 3.00 (p#0.001), 3.61 (p#0.001) and 3.89 (p#0.001) for people reporting two, three, four, five and six poor lifestyle behaviors, compared to people with 0–1 poor lifestyle behaviors. For Index 2, the Prevalence Ratio (p value) of poor self-rated health was 2.26 (p = 0.007), 3.29 (p#0.001), 4.68 (p#0.001), 6.48 (p#0.001), 7.91 (p#0.001) and 8.55 (p#0.001) for people reporting two, three, four, five, six and seven poor lifestyle behaviors, compared to people with 0–1 poor lifestyle behaviors. Associations between the combined lifestyle behavior index and frequent unhealthy days were statistically significant and similar to those observed for poor self-rated health. Conclusions: Engaging in a greater number of poor lifestyle behaviors was associated with a higher prevalence of poor HRQOL. This association was exacerbated when sleep quality was included in the index.