The ability to noninvasively detect the presence of species and assess physiological health by DNA and hormone analysis makes scat a valuable tool for ecology and conservation. We assessed factors associated with DNA and hormone degradation in a four-season study that employed detection dogs to collect scats from maned wolf (Chrysocyon brachyurus) in the Brazilian Cerrado, a tropical savanna landscape mosaic. Fecal DNA sample viability was assessed by attempting polymerase chain reaction amplification of a mitochondrial DNA (mtDNA) locus (∼ 246 bp) and a nuclear DNA zinc finger protein gene (∼ 195 bp). We assessed how extraction method, environmental exposure, and amount of odor, moisture and diet items in the sample influenced DNA amplification and allelic dropout rates. Samples that amplified mtDNA were assayed for glucocorticoids and thyroid hormone. Amount of odor and moisture (indicating freshness) predicted mtDNA amplification success, as well as mean hormone levels. While factors related to sample condition were negatively correlated with lower mean hormone levels, samples comprised mainly of fruit had higher levels of glucocorticoids and lower levels of thyroid hormone, and we thus interpret this result as biologically meaningful. In summary, DNA and hormone degradation are predicted by measures of sample freshness, making the assessment of sample quality an important criterion for sample collection as well to manage measurement error in analyses of hormone concentration associated with environmental disturbance.