The present study verified the efficacy of a dichloromethane Acmella oleracea (L.) R. K. Jansen extract against semi-engorged R. sanguineus s. l. female ticks and the effects of different concentrations on the germ cells and midgut epithelium of these ectoparasites through morphological and histological analyses. In vitro bioassays were performed (adult immersion test). For the cytotoxic evaluations, the semi-engorged females were allocated into five groups. The findings indicate that the dichloromethane A. oleracea extract was highly efficient against semi-engorged R. sanguineus s. l. females at concentrations higher than 10 mg/mL. The best results were obtained at 25 mg/mL, with an efficacy rate of 95%. The cytotoxic tests indicate that assessed the A. oleracea extract caused extensive damages to germ cells and the midgut epithelium of R. sanguineus s. l. ticks. The treatment affected the fertility of these ectoparasites, reducing their capacity to produce viable eggs and form the embryo and, consequently, to develop new individuals. Additionally, the treatment with the extract fraction affected the ectoparasites’ nutrition, which could delay or impair their development, consequently decreasing their mobility to find a suitable host and preventing them from completing their life cycle and moving to the following instar. Thus, the dichloromethane A. oleracea extract is a promising and viable alternative to control R. sanguineus s. l. ticks