This study investigated the potential relationship between shiftwork and work-related physical assault (PA) against nurses who are at high risk of violence globally. Nurses (6,300), randomly selected from the licensing database and working in Minnesota, were surveyed regarding PA experiences. Through a nested case-control study, nurses who reported a PA in the previous 12 months and controls who were randomly selected from their assault-free working months, respectively, identified exposures experienced during the month prior to the assault month (cases) and the random non-PA months (controls). Comparing case and control exposures, shiftwork was examined relevant to PA. Among 310 cases and 946 controls, most worked 8 hours or less (87%, 88%) during day shifts (44%, 70%). Multivariable analyses (odds ratios [ORs] and 95% confidence intervals [CIs]) revealed increased risk of PA for nurses working evening (OR = 1.55, 95% CI = [1.05, 2.27]), night (OR = 3.54, 95% CI = [2.31, 5.44]), and rotating day and evening (OR = 2.88, 95% CI = [1.22, 6.80]) shifts, which provides a basis for intervention opportunities.