This study aimed to investigate the association between poor sleep and risk of low-back pain (LBP) in healthcare workers. Using a prospective cohort design with 1-year follow-up, a total of 1955 healthcare workers (60% nurses) from 389 departments at 19 hospitals responded to questionnaires containing items related to lifestyle, health, and working environment. Associations between sleep scores (0–100) at baseline and LBP intensity (0–10) at follow-up were modelled using cumulative logistic regression accounting for clustering at the department level and adjusted for lifestyle and psychosocial confounders. In the full population of healthcare workers, 43.9% and 24.4% experienced moderate and poor sleep, respectively. In the fully adjusted model with good sleep as reference, moderate, and poor sleep increased the risk of LBP at follow-up, with odds ratios (OR’s) of 1.66 (95% confidence interval (CI) 1.35–2.04) and 2.05 (95% CI 1.57–2.69), respectively. Three sensitivity analyses including healthcare workers free from LBP, nurses, and nurses free from LBP at baseline, respectively, yielded similar results. In conclusion, poor sleep constitutes a potent risk factor for LBP among healthcare workers. The presented results provide strong incentives to evaluate and weigh current prevention policies against an updated biopsychosocial framework.