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PURPOSE Guatemala, a low- and middle-income country, has some of the poorest worldwide health indicators, including a high cancer incidence. We explored general cancer knowledge and breast and cervical cancer screening practices among Indigenous women in Lake Atitlán, Guatemala. METHODS We conducted 14 in-person, semistructured interviews and one focus group with 5 traditional midwives (comadronas). All participants were Mayan women from a rural community in Lake Atitlán (median age, 43.5 years; range, 18-72 years). The interviews and focus group were conducted in the participant’s preferred language, Spanish or Kaqchikel. Three coders analyzed the data using a process of inductive thematic analysis. RESULTS Participants had little knowledge and awareness about cancer in general, including screening and prevention. They associated the term cancer with death. Most had more knowledge about cervical cancer risk factors and screening compared with breast cancer. Participants voiced misinformation that breast cancer is caused by bad breast milk and that screenings for cervical cancer involve a hysterectomy. The terms uterine, ovarian, and cervical cancer were used interchangeably. None of the participants had ever been screened for breast cancer and all participants reported not knowing anyone who had been screened in the community. Some participants reported having received pap smears in the past. Major barriers reported include the cost and travel to health institutions and fear that the procedures may hurt. There was a strong preference for female doctors to conduct screenings given stigma. Participants reported experiencing discrimination and language barriers when attending hospitals. Participants mostly relied on family members and natural traditional medicine when they felt sick. Community and family support seemed to be key for health-seeking behaviors. CONCLUSION Findings highlight the lack of knowledge, limited screening practices, and numerous barriers among Mayan women in Guatemala. Efforts should focus on health promotion through educational workshops and increased accessibility to health services.