A few diatom species produce toxins that affect human and animal health. Among these, members of the Pseudo-nitzschia genus were the first diatoms unambiguously identified as producer of domoic acid, a neurotoxin affecting molluscan shell-fish, birds, marine mammals, and humans. Evidence exists indicating the involvement of another diatom genus, Amphora, as a potential producer of domoic acid. We present a strategy for the detection of the diatom species Amphora coffeaeformis based on the development of species-specific oligonucleotide probes and their application in microarray hybridization experiments. This approach is based on the use of two marker genes highly conserved in all diatoms, but endowed with sufficient genetic divergence to discriminate diatoms at the species level. A region of approximately 450 bp of these previously unexplored marker genes, coding for elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), was used to design oligonucleotide probes that were tested for specificity in combination with the corresponding fluorescently labeled DNA targets. The results presented in this work suggest a possible use of this DNA chip technology for the selective detection of A. coffeaeformis in environmental settings where the presence of this potential toxin producer may represent a threat to human and animal health. In addition, the same basic approach can be adapted to a wider range of diatoms for the simultaneous detection of microorganisms used as biomarkers of different water quality levels.