In this paper, we describe the development of a culture-based biochip for detecting mycobacteria in environmental samples. The biochips use the paraffinophilic nature of mycobacteria to rapidly detect and differentiate them from non-target microorganisms. New methods of depositing and patterning paraffin were developed to fabricate prototype biochips. Biochips were experimentally tested to demonstrate rapid detection of mycobacteria in environmental samples collected from a municipal sewage treatment plant. Our successful demonstration of the culture-based biochip technology presents an alternative approach for developing new technology to track microorganisms in complex environmental samples.