Rapid detection and viability assessment of pathogenic microorganisms, without the need for pre-enrichment steps, is critical in clinical microbiology, food safety, environmental quality assessment, and biosecurity. We demonstrate a powerful analytical concept and the related platform that enable in situ rapid detection, separation, sensitive quantification, and viability assessment of targeted microorganisms (bacteria and fungi) from minimally processed samples. This is based on a novel integration of magneto-affine selection and electrical impedance assay. The entire process, from capture to measurement, is executed using controlled magnetic fields to manipulate magnetic particles (MPs)-microbe affinity-based clusters, in a compact, portable setup equipped with cost-effective, single-use chambers. The system was tested for non-invasive in situ evaluation of model bacteria (Escherichia coli) and fungi (Saccharomyces cerevisiae) within clinically relevant concentration ranges, and it was demonstrated amenable for both commercial and custom MPs, proving its high versatility. The high capture efficiency, the ability to provide analytic results within 30 min directly from unprocessed samples (buffer and synthetic urine), and the high sensitivity in distinguishing live and dead cells in dynamic exposures represent significant advancements over existing assays and recommend the system as a screening tool for pathogen presence and antimicrobial susceptibility in clinical and environmental samples.