Abstract The study was carried out to assess the knowledge and practice of HIV positive mothers towards infant feeding options at Serere Health Center IV, Serere District. The study design was cross sectional and descriptive and it employed both quantitative and qualitative data collection methods. The study involved a total of 30 respondents. In conclusion, HIV positive mothers were not fully knowledgeable about infant feeding options as only half of the respondents 15 (50%) had heard about infant feeding options for HIV positive mothers and obtained information about infant feeding options from health workers. However, most 17 (56.7%) were not knowledgeable about the available mixed feeding options and 16 (53.3%) respondents were not aware of the importance of infant feeding options which was perhaps not surprising as most 17 (57%) had never been sensitized by health workers about infant feeding options. Respondents also had poor practices towards the use of infant feeding options and most 13 (43.3%) chose their current feeding option because it was cheap, 11 (36.7%) who said it was readily accessible while 6 (20%) said it was culturally appropriate. However, the majority of respondents 25 (83.3%) were laughed at or criticized for using infant feeding options by 12 (48%) friends, 7 (28%) community members and 6 (24%) relatives yet 25 (83.3%) reported that fear of being laughed at prevented use of using infant feeding options. Furthermore, all respondents 30 (100%) reported facing challenges in using infant feeding options including 14 (46.7%) lack of support by family members and friends, 10 (33.3%) baby not feeding well and 6 (20%) unnaturalness of some methods. The key recommendations included national sensitization programs about MTCT of HIV as well as how this could be reduced and prevented through adequate and effective use of replacement infant feeding options, supporting health facilities offering EMTCT programs with subsidized infant feeding options such as formula in an effort to improve uptake and utilization of this safe replacement infant feeding option.