As a part of a larger qualitative study to understand HIV-risk factors and impacts on people living with HIV (PLHIV) (52 women and 40 men) in Belu and Yogyakarta, Indonesia, this paper reports the influences of cultural practices and religious beliefs on sexual relationships and behaviours of participants as contributors for HIV transmission. This study was conducted from June to December 2020. Data collection was conducted using one-one-one in-depth interviews. Participants were recruited using the snowball sampling technique. Data analysis was guided by a qualitative data analysis framework. The findings showed that cultural practices in Belu related to the use of bride wealth, managing spousal disputes, marriage, and condom use, influenced spousal relationships and sexual behaviours or practices which contributed to HIV transmission. Javanese cultural practices and expectation of an ‘ideal wife’, Islamic religious beliefs about expected husband-wife relationships, forbidden premarital sex, and the participants’ individual interpretation of their religious beliefs about condom use spousal sexual relations, also influenced spousal sexual relations and behaviours, which supported HIV transmission among the participants. The findings indicate the need for HIV education programs that address cultural practices and religious beliefs for community members and population groups to enhance their understanding about HIV, condom use, and how cultural practices and religious beliefs play a role in HIV transmission. The findings also indicate the need for involvement of religious leaders in HIV education programs to bring insights to people and help them interpret their religious beliefs in health promoting ways. Future studies that explore different aspects of culture and religion which may contribute to HIV transmission are recommended.