The diagnosis of infectious diseases mostly relies on PCR-based analytical methods that, although consolidated and accurate, are strictly constrained to specialized personnel and dedicated laboratories, due to the complex procedures and the system architectures required. This limits the urgent expansion of the diagnostics towards massive and decentralized applications in countries lacking appropriate lab and health assistance. The PCR-free biosensing represents the most appealing solution since it provides the detection of an infectious pathogen without its genetic material amplification by PCR, thus, overcoming the technological limitations of the conventional diagnostics. In this contribution we present three electrochemical PCR-free biosensing strategies for the selective detection of pathogens, including severe acute respiratory syndrome coronavirus (SARS-CoV)-2, Pseudomonas aeruginosa and Leishmania infantum. The detection is based on the capacitive quantification, through electrochemical impedance spectroscopy (EIS), of the three pathogens’ nucleic acids (NAs) interacting via cooperative hybridization with complementary oligonucleotide probes anchored on top of commercial gold electrodes. The strategies allowed to directly reveal and quantify the target genomes without amplification and with a high level of selectivity.