The study was undertaken to evaluate the protective effect of Eupatorium perfoliatum (EP) 30C on the incidence of dengue fever. This study was designed as a prospective, open label, parallel cohort study. Participants were enrolled in two medicine cohort (MC) communities and one control cohort (CC) community. Participants in MC who were more than 5 years of age received four medicated globules and those aged between 1 and 5 years received two medicated globules of EP 30C once a week for 10 weeks. Participants in CC received no medical intervention. Both cohorts received information and educational material regarding dengue. The primary outcome was incidence of dengue cases based on laboratory confirmation or the clinical definition of dengue as per the case definition notified by the National Vector-Borne Disease Control Program, Government of India, during the 14 weeks of intervention and observation. The analysis included 20,607 participants residing in three slums of Delhi, of which MC and CC included 15,298 and 5,309 participants respectively. The overall protective effect of EP 30C in MC against probable/laboratory-confirmed dengue was 59.59% (95% confidence interval [CI], 50.74 to 66.85, p = 0.0001). The overall protective effect of EP 30C against laboratory-confirmed dengue was 72.78% (95% CI, 54.45 to 83.74, p = 0.0001): 26 cases per 15,298 (0.13 per 1,000 person-weeks) in MC versus 33 cases per 5,309 (0.46 per 1,000 person-weeks) in CC. The use of EP 30C was associated with some protection against probable and laboratory-confirmed dengue.