We describe the protocol for an inexpensive and nondestructive optical reflectance assay for the measurement of biofilm formation. Reflectance data are obtained using an Ocean Optics (Dunedin, Florida) USB 2000 spectrometer with a polychromatic light source. A fiber optic cable is used both for illumination and collection, and Ocean Optics OOIBase32 Platinum software is used for preliminary processing of the data. Differences in reflectance data collected at times ranging from 2 to 24 h distinguish between cell attachment and volume growth for two strains of Enterococci. Confocal scanning laser microscopy imaging is used to confirm these results. Phase contrast microscopy images are also obtained in conjunction with reflectance measurements for several different biofilm specimens. The experiments consider biofilm formation on glass and polystyrene substrata, but the method can be used for many other abiotic substrata of interest, both opaque and nonopaque.