The quantitative evaluation of bacterial populations is required in many studies, particularly in the field of microbiology. The current techniques can be time-consuming and require a large volume of samples and trained laboratory personnel. In this regard, on-site, easy-to-use, and direct detection techniques are desirable. In this study, a quartz tuning fork (QTF) was investigated for the real-time detection ofE. coliin different media, as well as the ability to determine the bacterial state and correlate the QTF parameters to the bacterial concentration. QTFs that are commercially available can also be used as sensitive sensors of viscosity and density by determining the QTFs’ damping and resonance frequency. As a result, the influence of viscous biofilm adhered to its surface should be detectable. First, the response of a QTF to different media withoutE. coliwas investigated, and Luria–Bertani broth (LB) growth medium caused the largest change in frequency. Then, the QTF was tested against different concentrations ofE. coli(i.e., 102–105colony-forming units per milliliter (CFU/mL)). As theE. coliconcentration increased, the frequency decreased from 32.836 to 32.242 kHz. Similarly, the quality factor decreased with the increasingE. coliconcentration. With a coefficient (R) of 0.955, a linear correlation between the QTF parameters and bacterial concentration was established with a 26 CFU/mL detection limit. Furthermore, a considerable change in frequency was observed against live and dead cells in different media. These observations demonstrate the ability of QTFs to distinguish between different bacterial states. QTFs allow real-time, rapid, low-cost, and non-destructive microbial enumeration testing that requires only a small volume of liquid sample.