Although acaricide chemotherapy is widely used to control tick infestation in Ethiopia, its effectiveness is uncertain due to misusage by herdsmen. Currently, there is no study being conducted in the South Omo Zone of Ethiopia which shows the knowledge, attitude, and practice (KAP) and associated factors of acaricide usage by herdsmen. Therefore, this study was conducted to assess KAP of 120 (83 male and 37 female) pastoralist and agro-pastoralist of Bena-Tsemay district through structured questionnaire survey. Accordingly, Ivermectin was the most preferred acaricide by majority (62.5%) of the herdsmen. Half (50%) of the herdsmen confessed that price of acaricide is the defining variable for acaricide preference in their location where 60.83% of them obtain acaricides from private drug shops. Majority (60%) of the respondents said that they obtain information about acaricide usage from drug sellers in the vet drug shops. According to 72.50% of the respondents, acaricide application/injection on the infested herd was conducted by the herdsmen. A 95.83% of our interviewee revealed that there was no training or awareness creation being given on how to inject or apply acaricide on tick infested animals. Moreover, all responders (100%) confessed that they didn't have a practice of weighing animals and measuring acaricide dosage prior to injection/application. The incidence of acaricide poisoning on animal and personnel was reported by 19.17% and 22.5% of respondents, respectively. Simple logistic regression analysis revealed that gender (OR = 5.09, OR 95% CI = 2.30–11.72), practice of acaricide rotation (OR = 3.22, OR 95% CI = 1.41–7.64) and personnel preference for acaricide application (OR = 2.66, OR 95% CI = 1.18–6.15) were significantly (P < 0.05) associated with the knowledge score of the respondents. On the other hand, respondent's attitude score was significantly (P < 0.05) associated with their acaricide rotation practice (OR = 3.20, OR 95% CI = 1.39–7.53) and personnel preference for acaricide application (OR = 6.61, OR 95% CI = 2.78–16.93). Similarly, practice of acaricide rotation (OR = 5.31, OR 95% CI = 2.26–12.96) and personnel preference for acaricide application (OR = 7.21, OR 95% CI = 3.03–17.99) were significantly linked with the practice score of the respondents towards acaricide usage. In conclusion, ticks are the major challenge in the study area despite widespread usage of acaricides. Because of extensive misusage of available acaricides, awareness creation should be applied to narrow KAP gaps and to conserve the efficacy of these chemicals. Furthermore, acaricide efficacy investigation (in vitro and in vivo) should be conducted to know the status of commonly used acaricides in the area.