Rationale: The overuse of antibiotics has been an important clinical issue, and antibiotic exposure is linked to alterations in gut microbiota, which has been related to risks of various chronic diseases such as cardiovascular disease and cancer. Also, duration of antibiotic exposure may be a risk factor of premature death. Objective: We investigated associations of life-stage and duration of antibiotic use during adulthood with risks of all-cause and cause-specific mortality. Methods and Results: This prospective cohort study included 37 516 women aged ≥60 years who were free of cardiovascular disease or cancer from the Nurses' Health Study. Participants reported a total amount of time they used antibiotics (none, <15 days, 15 days to <2 months, or ≥2 months) in the middle- (age, 40-59) and late adulthood (age, 60 or older). We estimated hazard ratios for all-cause mortality and deaths from cardiovascular disease or cancer over 10 years according to duration of antibiotic use. During 355 918 person-years of follow-up, we documented 4536 deaths from any cause (including 728 cardiovascular deaths and 1206 cancer deaths). As compared with women who did not use antibiotics, those who used them for ≥2 months in late adulthood had increased risks of all-cause mortality (hazard ratio, 1.16 [95% CI, 1.01-1.33]) and cardiovascular mortality (hazard ratio, 1.49 [95% CI, 1.04-2.13]), but not cancer mortality (hazard ratio, 0.85 [95% CI, 0.65-1.12]) after adjustment for chronic metabolic diseases, antibiotic use during middle adulthood, indication for use, demographic factors, and lifestyle/dietary factors. The association was more evident among women who also used antibiotics in middle-adulthood than among those who did not use during this life-stage. Conclusions: Long-term use of antibiotics in late adulthood may be a risk factor for all-cause and cardiovascular mortality. The unfavorable effect of antibiotic exposure for subsequent risks of deaths due to chronic diseases needs to be considered.