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Despite the widespread use of anaerobic soil disinfestation (ASD) to improve soil health, its impact on soil antibiotics, human pathogenic bacteria (HPB) and antibiotic resistance genes (ARGs) remains unclear. To address this knowledge gap, we conducted an incubation study in manure-amended soil with three treatments: no treatment (CK), crop straw only (CS), and CS combined with water flooding and plastic covering (ASD). The results showed that ASD increased the concentrations of tetracycline by 27 % and β-lactam by 22 %, while depleting macrolide by 51 %. Furthermore, ASD increased the numbers of potential HPB species and genes by 4.1 % and 23 %, respectively. Co-occurrence network analysis demonstrated intensified cooperation and competition among HPB and HNPB (human non-pathogenic bacteria) species under ASD, favoring the prevalence of HPB species such as Escherichia coli , Streptococcus equi , Clostridium tetani , and Bacillus anthracis. Potential HPB species in Proteobacteria exhibited negative correlations with macrolide and β-lactam antibiotics, whereas those in Firmicutes showed positive correlations with tetracycline antibiotics, suggesting that specific bacterial taxa contributed to the production and degradation of antibiotics in response to ASD. Interestingly, CS mitigated the presence of ARGs in soil, while ASD further amplified this reduction, almost eliminating nalC , acrB , OXA-60 , VIM-7 , tetL , and tetQ. The variation in ARG profiles (91.7 %) could be explained by antibiotics, HPB, and HNPB species, indicating their collective contribution to reducing soil ARGs under ASD. Moreover, host prediction and network analysis revealed that, compared with CK, where potential HPB species were the main carriers of ARGs, soil ARGs were less frequently carried by potential HPB species after ASD, indicating a reduction in the collaborative dissemination of HPB and ARGs in manured soil. In conclusion, ASD provides a promising strategy for mitigation of soil biologic contamination, which can have profound significance for environmental safety and agricultural sustainability. • ASD altered soil distribution of tetracycline, β-lactam and macrolide antibiotics. • ASD enriched soil potential HPB species by enhancing connections between HPB and HNPB species. • ASD removed most of the common ARGs, which was weakly correlated with soil antibiotics. • Most potential HPB species boosted by ASD were not the hosts of ARGs in soil. • ASD decreased the risks of synchronous dissemination of HPB and ARGs in soil. [ABSTRACT FROM AUTHOR]