Bacteriophage MooMoo is a temperate phage that was enriched from a water sample using Mycobacterium smegmatis as the host. Based upon published observations, we hypothesized that MooMoo may encode genes that are toxic to Mycobacterium smegmatis, when overproduced. Five genes (gp87, g88, gp89, gp90, and gp91) were tested for toxicity because they encode small proteins that have no known function. Each gene was amplified from the phage genome by polymerase chain reaction (PCR) and cloned into an Escherichia coli/M. smegmatis shuttle/expression vector. The recombinant plasmids were propagated in E. coli, verified by PCR, then moved into M. smegmatis cells. The cloned genes were induced and cell growth was monitored on plates. The toxicity of each gene was scored on a Toxicity Index (TI) from 0–5, with 0 corresponding to no toxicity (abundant growth) and 5 representing the strongest toxicity (no growth). MooMoo gp87 displayed the strongest toxicity and was chosen for further analysis. To determine how gp87 interferes with M. smegmatis growth, we performed a protein-protein interaction assay using a bacterial two-hybrid screen. This analysis identified an interaction between gp87 and the host metabolic machinery. Future experiments include modeling the interacting surfaces of the proteins and attempting to isolate host mutants that suppress the toxicity. Our analysis represents a general approach for elucidating gene function and may identify potential new targets for therapeutics.