We characterize bacteria of the gastrointestinal tract of 28 Northern Saw-whet Owls (Aegolius acadicus) netted on the Mohonk Preserve near New Paltz, New York, during autumn of 2017. We used selective culture media to determine the presence and assess the point infection level of Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., and Staphylococcus aureus in the buccal, cloaca, feces, and pellets of Northern Saw-whet Owls. We tested for associations between bacteria type, sample point location, and infection levels and then assessed the influence of bacterial infection on the health of owls using multivariate analysis of mass/wing chord body condition index estimates and heterophil to lymphocyte ratios. All Northern Saw-whet Owls tested positive for at least one type of bacteria. We found an inverse relationship between the number of bacteria types detected per individual and the percentage of Northern Saw-whet Owls affected, with 46.43%, 32.14%, and 3.57% of individuals showing presence of 2, 3, and 4 types of bacteria, respectively. Multivariate analysis revealed a significantly heightened presence of S. aureus in the buccal cavity, and Spearman's rho analysis revealed positive correlations between site colonization levels and bacterial species levels. We found no significant relationship between bacterial load and mass/wing chord body condition indexes or heterophil to lymphocyte ratios. We did, however, find a positive relationship between heterophil to lymphocyte ratios and Leucocytozoon infections. Providing baseline information of common microflora can help future research recognize potentially harmful gastrointestinal pathogens. Therein, as the first study to characterize bacteria of the Northern Saw-whet Owl, this research adds to the information portfolio of the species and may benefit future research efforts.