ObjectiveTo compare the differences between clinical and environmental sources of Acinetobacter baumannii （AB） at a tertiary hospital in Guangzhou in terms of molecular epidemiological characteristics， antimicrobial resistance， disinfectant tolerance， virulence and the distribution of related genes， as well as plasmid epidemiological characteristics； and to assess the association between environmental and clinical strains and the potential transmission risks associated with environmental contamination. MethodsA total of 118 clinical and 102 environmental AB isolates were collected from a tertiary hospital in Guangzhou between 2024 and 2025. Species identification was performed using mass spectrometry and 16S rRNA sequencing； minimum inhibitory concentrations （MICs） for eight antimicrobial agents were determined using the agar dilution method； biofilm formation capacity was assessed using the crystal violet staining method； A Galleria mellonella infection model was used to evaluate the virulence； whole-genome sequencing was employed for multilocus sequence typing （MLST）， phylogenetic analysis， and studies on the distribution of resistance， disinfectant resistance and virulence genes， and plasmid screening. ResultsA total of 38 known ST types were identified among the 220 AB strains， with ST2 and ST40 being the predominant lineages. Antimicrobial susceptibility testing revealed that the clinical infection group exhibited resistance to minocycline， imipenem and other antibiotics， as well as the tolerance levels to the disinfectants triclosan and benzalkonium bromide， were generally higher than those in the environmental group （P＜0.001）， and the detection rates of drug resistance genes such as bla_OXA-23 and adeB all exceeded 70.0%. Statistical analysis confirmed that the MIC values of TCS and BAB were significantly positively correlated with various antibiotic resistance phenotypes （P＜0.05）. In biofilm experiments， the proportion of strong biofilm-forming strains in the environmental group （64.7%） was significantly higher than that in the clinical infection group （11.9%；χ²=69.06， df=2， P＜0.000 1）， and the mortality rate of the strains increased with enhanced biofilm-forming ability. Furthermore， a total of 41 plasmid lineages and 219 insertion sequences were detected； ISs such as IS1006 and IS1007 were frequently co-occurring with resistance and virulence genes including bla_OXA-23 and katA. ConclusionsThe predominant lineages of AB in this tertiary hospital were ST2 and ST40. The clinical infection group generally exhibits higher levels of antibiotic resistance and tolerance to disinfectants compared to the environmental group， while biofilm-forming ability differs ： the environmental group dominates in strong biofilm formation， and the clinical group dominates in weak to moderate biofilm formation. Plasmids and ISs may be involved in the co-occurrence and dissemination control strategies and for the molecular epidemiological surveillance of multidrug-resistant bacteria.