Healthcare workers and patients may acquire infections during the provision and acquisition of healthcare services, respectively, due to exposure to microorganisms in healthcare facilities. Studies have shown that compliance with standard infection prevention guidelines is low among healthcare workers. This study investigated the factors influencing compliance with infection prevention and control guidelines among healthcare workers in Seme Sub-County. Data was collected using an observational checklist, questionnaires and key informant interviews. Quantitative data was analyzed using descriptive statistics, and logistic regression, using SPSS (v.25) while qualitative data was subjected to content analysis. A total of 109 healthcare workers from all the healthcare facilities in Seme Sub-County participated in the study. About 20% of the facilities had written infection prevention and control policy guidelines. County and sub-county hospitals and only one health center had infection prevention and control committee, while none of the dispensaries had an infection prevention and control committee in place. Majority of the health care workers reported a good uptake of IPC domains; hand washing (80.7%), waste segregation (94.5%) and injection safety (84.9%), although upon observation, only a small proportion of healthcare facilities followed the infection prevention and control domains. Majority of staff mentioned lack of training on infection prevention and control practices, and the major barriers to compliance included lack of dissemination of guidelines, inadequate equipment and supplies, and ignorance among healthcare workers. County level of healthcare facility was significantly associated with good uptake of hand washing (p=0.04), while nurses were more likely to have good uptake of hand washing and waste segregation compared to medical officers (p=0.04). These findings suggest need for improvement on infection prevention and control within the facilities. It is necessary for the hospital administration to reactivate infection prevention and control committees in all major facilities, and conduct regular audits and in-service training to enhance good compliance and implementation of infection prevention and controlin Seme Sub-County.