Rationale: Although the mainstay of sepsis treatment is timely initiation of broad-spectrum antimicrobials, treatment delays are common, especially among patients who develop hospital-onset sepsis. The time of day has been associated with suboptimal clinical care in several contexts, but its association with treatment initiation among patients with hospital-onset sepsis is unknown. Objectives: Assess the association of time of day with antimicrobial initiation among ward patients with hospital-onset sepsis. Methods: This retrospective cohort study included ward patients who developed hospital-onset sepsis while admitted to five acute care hospitals in a single health system from July 2017 through December 2019. Hospital-onset sepsis was defined by the Centers for Disease Control and Prevention Adult Sepsis Event criteria. We estimated the association between the hour of day and antimicrobial initiation among patients with hospital-onset sepsis using a discrete-time time-to-event model, accounting for time elapsed from sepsis onset. In a secondary analysis, we fit a quantile regression model to estimate the association between the hour of day of sepsis onset and time to antimicrobial initiation. Results: Among 1,672 patients with hospital-onset sepsis, the probability of antimicrobial initiation at any given hour varied nearly fivefold throughout the day, ranging from 3.0% (95% confidence interval [CI], 1.8-4.1%) at 7 a.m. to 13.9% (95% CI, 11.3-16.5%) at 6 p.m., with nadirs at 7 a.m. and 7 p.m. and progressive decline throughout the night shift (13.4% [95% CI, 10.7-16.0%] at 9 p.m. to 3.2% [95% CI, 2.0-4.0] at 6 a.m.). The standardized predicted median time to antimicrobial initiation was 3.2 hours (interquartile range [IQR], 2.5-3.8 h) for sepsis onset during the day shift (7 a.m.-7 p.m.) and 12.9 hours (IQR, 10.9-14.9 h) during the night shift (7 p.m.-7 a.m.). Conclusions: The probability of antimicrobial initiation among patients with new hospital-onset sepsis declined at shift changes and overnight. Time to antimicrobial initiation for patients with sepsis onset overnight was four times longer than for patients with onset during the day. These findings indicate that time of day is associated with important care processes for ward patients with hospital-onset sepsis. Future work should validate these findings in other settings and elucidate underlying mechanisms to inform quality-enhancing interventions.