A major concern with medical and dental biomaterials is colonization of these materials with microbial biofilms. One material processed using chemical vapor deposition and other conventional top-down nanomanufacturing technologies that has recently been considered for use in preventing growth of microorganisms is the nanocrystalline diamond. Nanocrystalline diamond coatings have been evaluated for use as coatings on medical implants (e.g., hip prostheses) and surgical tools due to their low coefficient of friction, high corrosion resistance, high hardness, and high wear resistance. In this study, the microstructural properties and microorganism interaction behavior of nanocrystalline diamond coatings were examined. A device for examining microbial biofilms known as a CDC biofilm reactor was used to examine the interaction between a fluorescent microorganism, Pseudomonas fluorescens, and nanocrystalline diamond coatings in a continuous perfusion environment. Biofilm formation was evident on the nanocrystalline diamond surface after 24 h. No correlation between grain size or morphology and cell density was observed; large variations in P. fluorescens growth on the coatings were observed, even for the samples with similar grain sizes and morphologies. The results of this study suggest that nanocrystalline diamond coatings do not prevent Pseudomonas fluorescens biofilm development in a continuous perfusion environment. Additional treatment of the nanocrystalline diamond coatings with antimicrobial and/or antifouling agents would be necessary to prevent formation of microbial biofilms. The development of novel continuous flow technologies for evaluating the growth of microbial biofilms on biomaterials will provide a better understanding of biomaterial-microorganism interaction and will enable the creation of enhanced antimicrobial biomaterials.