Methicillin-resistant (MRSA) is a leading cause of life-threatening endovascular infections. Endothelial cell (EC) damage is a key factor in the pathogenesis of these syndromes. However, genetic factors related to the EC damage have not been well studied. This study aims to identify genetic determinants that impact human EC damage by screening the genome-wide Nebraska Transposon Mutant Library (NTML). A well-established MTT assay was used to test the in vitro damage of human EC cell line (HMEC-1) caused by each mutant strain in the NTML. We first confirmed some global regulators and genes positively impact the EC damage, which is consistent with published results. These data support the utility of the high-throughput approach. Importantly, we demonstrated 317 mutants significantly decreased the EC damage, while only 6 mutants enhanced the EC damage vs. parental JE2 strain. The majority of these genes have not been previously defined to affect human EC damage. Interestingly, many of these newly identified genes are involved in metabolism, genetic and environmental information processing, and cellular processes. These results advance our knowledge of staphylococcal genetic factors related to human EC damage which may provide novel targets for the development of effective agents against MRSA endovascular infection.