After isolation from a pulp mill wastewater treatment facility, two yeast strains, designated SPT1 and SPT2, were characterized and used in the development of mediated biochemical oxygen demand (BOD) biosensors for wastewater. 18S rRNA gene sequence analysis revealed a one nucleotide difference between the sequence of SPT1 and those of Candida sojae and Candida viswanthii. While SPT2 had the highest overall homology to Pichia norvegensis, at only 73.5%, it is clearly an ascomycete, based on BLAST comparisons and phylogenetic analyses. Neighbor-joining dendrograms indicated that SPT1 clustered with several Candida spp., and that SPT2 clustered with Starmera spp., albeit as a very deep branch. Physiological tests, microscopic observations, and fatty acid analysis confirmed that SPT1 and SPT2 are novel yeast strains. Physiological tests also indicated that both strains had potential for use in mediated biosensors for estimation of BOD in wastewater. The lower detection limits of SPT1- and SPT2-based K3Fe(CN)6-mediated biosensors for a pulp-mill effluent were 2 and 1 mg BOD/L, respectively. Biosensor-response times for effluents from eight different pulp mills were in the range of 5 min. Reliability and sensitivity of the SPT1- and SPT2-based biosensors were good, but varied with the wastewater.