The rapid and accurate identification of microbes is critical for a variety of industries, notably healthcare, bioterrorism/defense, food and agriculture, and environmental testing. Nucleic acid-based identification platforms, in particular, have introduced marked improvements in the overall specificity and sensitivity of pathogen detection. While tremendous technical progress has been made in addressing the specific demands of these various sectors, there still exists a significant unmet need for a rapid and universal microbial identification platform in the clinic. Using a set of universal, target-agnostic probes, microbial species can be readily distinguished from one another based upon the observed variability in the total number of unique hybridization events between each probe and each target genome. In this way, both the identity of the microbe and its infectious load can be determined. To that end, this work first establishes the efficacy of a specific universal-probe that builds off of existing toehold-probe technologies. Given the overly narrow thermodynamic constraints of single-mismatch protectors in traditional toehold-probes, and the inherent noisiness of standard molecular probes, we herein introduce “sloppy” or mismatch-tolerant universal toehold-probes, and validate their efficacy by demonstrating successful detection and characterization of viral subpopulations or quasi-species in patient-derived viral DNA. This work also investigates several novel schemes that utilize a set of target-agnostic universal toehold-probes to rapidly and accurately identify bacterial species with high sensitivity. These include probe-capture, endonuclease cleavage, size-exclusion chromatography, and fluorescence in situ hybridization.