Hospital-acquired infection (HAI) and antimicrobial resistance (AMR) represent major challenges in healthcare system. Despite numerous studies have assessed environmental and patient samples, very few studies have explored the microbiome and resistome profiles of medical staff including nursing workers. This cross-sectional study was performed in a tertiary hospital in China and involved 25 nurses (NSs), 25 nursing workers (NWs), and 55 non-medical control (NC). Stool samples from all participants and hand samples (i.e., the microbiome sample from hand skin, which were collected by swabbing both hands with a sponge-swab soaked with neutralized buffer and centrifuging the liquid buffer) from NSs and NWs were collected for metagenomic analysis. Metagenomic analysis revealed that medical staff exhibited lower abundances of beneficial species such as Blautia, and Bifidobacterium in the gut microbiome. However, an important potential pathogen, Staphylococcus haemolyticus, was enriched in the hands of NWs, suggesting a considerable prevalence of pathogenesis and multi-drug resistance. Accordantly, ARG analysis revealed worse hand hygiene among NWs than among NSs, characterized by a higher diversity of ARGs and a higher abundance of ARGs conferring multi-drug resistance including mdtF, acrB, AcrF and evgS. This study provides a comprehensive overview of the microbial and ARG profiles in the gut and hands of NSs and NWs. The higher abundance of potential pathogens and diverse multi-drug resistant ARGs in NWs hands indicates insufficient hand hygiene and a higher risk of HAI in this subgroup. This study is the first to highlight the critical need to improve hand hygiene among NWs, thus mitigating the risks of AMR and HAI.