In this paper, we describe the development of a culture-based biochip device for rapid detection of mycobacteria in environmental samples. Individual biochips rely upon the unique paraffinophilic nature of mycobacteria to rapidly and selectively adhere to the surface of the device. We used prototype biochips to experimentally demonstrate the concept of rapid and selective detection of mycobacteria by testing pure cultures and using epifluorescence microscopy to visualize microorganisms on the surface. As an alternative, rapid approach for identifying the biomass on the biochip surface, we used microwaves in the 10 to 26 GHz frequency range. The results of this study indicate that different microorganisms are responsible for specific shifts in resonance frequencies of a microwave cavity. By combing the semi-selective paraffin surface of the biochip with the microorganism-specific response to the microwaves, we have developed an improved analytical system with the potential to rapidly identify and enumerate mycobacteria in environmental samples in as little as 2 h.