Background: Healthcare-associated infections (HAIs) and antimicrobial resistance (AMR) have been becoming the public and global health issues. The purpose of this study is to estimate the direct economic burden attributable to HAIs and AMR.Methods: This study applied propensity score matching (PSM) methodology to conduct a prospective case-control study of direct disease burden attributable to HAIs and AMR 16th December, 2017 to 16th April, 2018 in a teaching hospital in Nepal. Clinical information was retrieved from Hospital Health Information and electronic medical record systems, as well as the microbiology lab system. The finance system was used to determine the health expenditure and length of hospitalization of HAIs, HAIs-AMR and Non-HAIs patients. STATA 12.0 was used to conduct descriptive analysis, (bivariate) χ2 test, paired/independent T test, PSM (B=0.25a, nearest neighbour 1:1 matching, General Boosted Model, GBM). The statistically significant level was set at P < 0.05.Results: HAIs patients and HAIs-AMR patients had statistically significant higher expenditures and longer length of hospital days than Non-HAI inpatients during the study period (P<0.05). The additional average total medical expenditure, medicines expenditure, out-of-pocket expenditure and length of hospitalization per patient caused by HAIs were 17,224.93 Rupees, 11,947.49 Rupees, 15,776.57 Rupees and 7 days, respectively. In the meantime, the extra total medical expenditure, medicines expenditure, out-of-pocket expenditure and length of hospitalization attributable to HAIs-AMR were 39,879.63 Rupees, 21,173.63 Rupees, 38,770.87 Rupees and 9 days, respectively. Moreover, the percentage of out-of-pocket expenditure accounting for total medical expenditure of HAIs Group was 94.24% while it was 96.75% of HAIs-AMR Group.Conclusions: It was the first time to apply the research framework of the previous study in China to estimate the direct economic burden caused by HAIs and AMR in a teaching hospital of Nepal. HAIs and AMR have substantially led to excess direct economic burden for patients and their family due to the low Gross Domestic Product (GDP) and low health expenditure in Nepal. This calls for active collaboration with developed countries to reduce the threat caused by HAIs and AMR to prevent the transnational communication.