Tree holes serve as breeding habitats for a variety of insects, however little is known about the microbial community found in these habitats. The goal of this study is to quantify and identify culturable bacteria from tree hole communities to provide an initial baseline of microbial communities. Water samples were collected from two tree holes in Westchester County, NY, every other week in September-October 2019. Serial dilutions were plated and individual colonies were counted. Isolates were tested against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa and producers were identified with PCR and Sanger sequencing. Time of collection and samples did not influence the number of CFU or percent producers, however 10 isolates demonstrated potential for antibiotic production against S. aureus. Future work should increase temporal sampling, as well as number of samples to determine tree or site level variation in bacteria numbers and antibiotic production.