Microbial infections contribute to ~20% of malignancy. Plasma-derived cell-free DNA presents a promising avenue for non-invasive cancer diagnostics, capturing microbial signatures. We analyzed 261 plasma from 50 patients with lung adenocarcinoma by targeted and whole exome sequencing at 10,000 × and 340 × depth, respectively. Comparative analyses of Kraken 2 and IPD2 reveal substantial discrepancies, highlighting challenges in microbial DNA quantification and the need for stringent bioinformatics approaches to ensure accurate cancer microbiome profiling.