Background: Papua New Guinea (PNG) is one of the 14 countries categorised as having a triple burden of tuberculosis (TB), multidrug-resistant TB (MDR TB), and TB-human immunodeficiency virus (HIV) co-infections. TB infection prevention and control (TB-IPC) guidelines were introduced in 2011 by the National Health Department of PNG. This study assesses the implementation of this policy in a sample of district hospitals in two regions of PNG. Methods: The implementation of TB-IPC policy was assessed using a survey method based on the World Health Organization (WHO) IPC assessment framework (IPCAF) to implement the WHO’s IPC core components. The study included facility assessment at ten district hospitals and validation observations of TB-IPC practices. Results: Overall, implementation of IPC and TB-IPC guidelines was inadequate in participating facilities. Though 80% of facilities had an IPC program, many needed more clearly defined IPC objectives, budget allocation, and yearly work plans. In addition, they did not include senior facility managers in the IPC committee. 80% (n=8 of 10) of hospitals had no IPC training and education; 90% had no IPC committee to support the IPC team; 70% had no surveillance protocols to monitor infections, and only 20% used multimodal strategies for IPC activities. Similarly, 70% of facilities had a TB-IPC program without a proper budget and did not include facility managers in the TB-IPC team; 80% indicated that patient flow poses a risk of TB transmission; 70% had poor ventilation systems; 90% had inadequate isolation rooms; and though 80% have personal protective equipment available, frequent shortages were reported. Conclusions: The WHO-recommended TB-IPC policy is not effectively implemented in most of the participating district hospitals. Improvements in implementing and disseminating TB-IPC guidelines, monitoring TB-IPC practices, and systematic healthcare worker training are essential to improve TB-IPC guidelines’ operationalisation in health settings to reduce TB prevalence in PNG.