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Background. People with HIV (PWH) exhibit a markedly increased risk of anal cancer, especially men who have sex with men (MSM), with specific microbiota signatures possibly contributing to greater human papillomavirus (HPV) oncogenic potential. Low specificity of the current screening strategy for detecting high-grade squamous intraepithelial lesions (HSIL) hinders anal cancer prevention. We investigated microbiome derivatives associated with HSIL in anal cytology samples. Methods. We recruited a discovery and a validation cohort at four clinical sites in Spain and Italy. Study participants were mostly MSM undergoing HSIL screening with high-resolution anoscopy and anal biopsies to confirm HSIL. We extracted the bacterial DNA, proteins, and metabolites from anal cytology samples, and we performed 16SrRNA gene sequencing, mass spectrometry, and targeted metabolite quantification. Results. We included 213 participants, 167 in the discovery cohort (70 with confirmed HSIL) and 46 in the validation cohort (25 with confirmed HSIL). While we did not find clear microbiome composition signatures associated with HSIL, the microbiome associated with HSIL overexpressed proteins involved in the production of succinyl-CoA and cobalamin, with levels consistently increased in subjects with HSIL in the discovery and validation cohorts. Combined measurement of succinyl-CoA and cobalamin overperformed anal cytology, improving sensitivity from 91.2% to 96.6%, specificity from 34.1% to 81.8%, positive predictive value from 48.1% to 77.8%, and negative predictive value from 85.3% to 97.3%. While anal cytology correctly classified only 59.9% of individuals, combined measurement of both metabolites improved the classification to 87.7%. This test overcame internal (adjusted AUC 0.877) and external validation. From 98 false-positive cytologic results, succinyl-CoA and cobalamin level measurement reclassified 49 (50%) to true negatives. Finally, we demonstrate greater in vitro production of succinyl-CoA and cobalamin in bacteria associated with HSIL or cancer vs. those presumably protective. Conclusions. Cobalamin and succinyl-CoA are overexpressed in the anal microbiome of patients with HSIL and show excellent diagnostic capacity. Their measurement overperforms anal cytology to screen for patients with HSIL. Hence, we discovered two powerful biomarkers for which detection methods can readily be established, that could improve the current strategy of anal cancer screening.