infection (CDI), is the most common healthcare problem primarily involving the colon of individuals who's gut microbiota has been disrupted. Proteobacteria (officially updated and recognized as Pseudomonadota), a minor gut-associated microbial community within a healthy host, could serve as a metric for CDI. However, the alterations of specific members of Proteobacteria in the context of CDI are not thoroughly understood. Based on the summary data of microbiome from 7,738 participants in the Dutch cohort, linkage disequilibrium score regression (LDSC) was used to explore the causal effect of 207 gut microbiome on CDI. Secondly, we performed a Mendelian randomization analysis to investigate the causal relationship between 31 microbiota taxa affiliated with Proteobacteria and CDI. Finally, three significant taxa ( < 0.05, OR > 1) were utilized to conduct the mediation analysis of 1,400 metabolites based on a two-step Mendelian randomization study (two-step MR). The inverse-variance weighted method was conducted as a primary analysis to estimate the causal effect, and the robustness of the results was tested via sensitivity analysis using multiple methods. Bivariate LDSC analysis identified a strong correlation between four populations affiliated with Proteobacteria (, Pasteurellales and ) and CDI. In two-step MR, Burkholderiales order exerted detrimental effects on CDI by decreasing the levels of 3-hydroxylaurate (OR 0.896; 95%CI, 0.803-0.998; = 0.047), indicating that metabolite did act as mediator between gut microbiota and CDI. We conducted a study to assess the relations between genetically predicted gut microbiota and metabolite levels with CDI. These results highlight the potential of targeting Burkholderiales and 3-hydroxylaurate as a new antimicrobial strategy against CDI.