An amplification-free, nanopore-based nucleic acid detection platform has been demonstrated for rapid, 16S rRNA sequence-specific detection of Neisseria gonorrhoeae at 10–100 CFU mL⁻¹ in human urine against background bacterial flora at 1000 CFU mL⁻¹. Gonorrhea is a very common notifiable communicable disease, antibiotic resistant strains have emerged, and the rate of reported gonococcal infections continues to increase. Since rapid clinical identification of bacterial pathogens in clinical samples is needed to guide proper antibiotic treatment and to control disease spread, it is important to engineer rapid, sensitive, selective, and inexpensive point-of-care (POC) diagnostic devices for pathogens such as N. gonorrhoeae. Our detector technology is based on straightforward conductometric detection of sustained blockage of a glass nanopore. Charge neutral, complementary peptide nucleic acid probes are conjugated to polystyrene beads to capture N. gonorrhoeae 16S rRNA selectively. In the presence of an electric field applied externally through a glass nanopore, the PNA-microbead conjugates that acquire substantial negative charge upon target hybridization are driven to the smaller diameter nanopore. At least partial blockage of the nanopore results in a sustained drop in ionic current that can be measured easily with simple electronics. The ability to detect N. gonorrhoeae over the range of 10 to 100 CFU mL⁻¹ spiked in human urine was demonstrated successfully with estimated sensitivity and specificity of ∼98% and ∼100%, respectively. No false positives were observed for the control group of representative background flora (E. coli, K. pneumoniae, and E. faecalis) at 1000 CFU mL⁻¹. Also, N. gonorrhoeae at 50 CFU mL⁻¹ was successfully detected against 1000 CFU mL⁻¹ of background flora in urine. These results suggest that this amplification-free technology may serve as the basis for rapid, inexpensive, low-power detection of pathogens in clinical samples at the POC.