Previously, we reported on the use of microelectromechanical systems (MEMS) fabrication technologies to develop paraffin surfaces for miniaturization of culture-based detection and rapid quantification of Mycolata in environmental samples. In the current study, the novel culture-based biochip was calibrated with a broad range of pure cultures of Mycolata including Gordonia spp. isolated from activated sludge foam. The biochip successfully recovered Gordonia amarae spiked into a sample of mixed liquor collected from a municipal activated sludge system. Comparisons of these results with molecular biology-based assays including 16S rRNA-targeted fluorescence in situ hybridization (FISH) and antibody staining demonstrated that the biochip provides a more rapid and user-friendly platform for reliable identification and quantification of Mycolata in full-scale municipal activated sludge sewage treatment plants. The results of this work successfully demonstrate an alternative platform technology for inexpensive monitoring of environmental microorganisms using existing expertise by potential users in the area of bacterial cultivation.