Background: Hospital-acquired infections (HAI) contribute to prolonged hospital stays and account for a substantial economic burden to healthcare systems. Middle-income countries (MICs) experience a greater burden of HAI than developed countries. Evidence on the cost-effectiveness of interventions to reduce HAI is required to inform decision-making in these settings. Aim: To synthesise the evidence on cost-effectiveness as related to HAI interventions in MICs and to assess the quality of this evidence. Methods: A systematic review of published literature on the cost-effectiveness of interventions to reduce the incidence of HAI in MICs between 2000 and 2018 was conducted. Results: Six studies met the pre-determined inclusion criteria. The studies were from three countries: Thailand; India; and Vietnam. The evidence suggests that interventions to reduce HAI are cost-effective and, in most cases, cost-saving to healthcare systems. The quality of the reporting varied across studies. Conclusions: The implementation of HAI prevention interventions appears to be a high value use of resources in MICs. There is a need for further cost-effectiveness analyses in a wider range of MICs in order to confirm these findings. Improved standardisation and quality of reporting is required.