To improve malaria control measures, taking into account local beliefs and practices are essential. In the present study, the PEN-3 culture model as a theoretical framework was employed to examine how health beliefs, behaviors and practices associated with malaria prevention in two communities, Afghani refugees and Bluchi residents in a malaria endemic area located in southeast of Iran. A mixed-methodology was designed by means of two quantitative surveys and qualitative focus groups. Cross-sectional survey using questionnaires combined with focus group discussions carried out by using a pre-coded questionnaire and eight qualitative focus groups were held. In total, 385 participants volunteered to take the cross-sectional survey, with 194 Afghanis, 191 Bluchis completing quantitative surveys and also 46 participated in the qualitative focus groups. Symptoms of malaria were the most frequently mentioned by both groups. A significant association between education level and knowledge on malaria transmission was also observed within both communities. Although the majority of respondents associated the disease transmission with mosquito bites only 16.5% Afghanis as compared to 63.4% Baluchis reported to use mosquito net. Data from focus group emerged three themes includes similarity in perception about malaria, difference in type of treatment and decision making and, finally resemblance to prevention of malaria in both communities. In the study, cultural differences in the recognition and interpretation of prevention and treatment of malaria within two communities were identified. Cultural match of Afghani and Baluchi perspective to malaria interventions and services will improve receptivity to, acceptance of, and salience of these efforts.