This experiment was carried out to determine the concentration of microbial load, identify and isolate a specific microbe (E. coli) on hatching eggs from the breeder house, comparing it to subsequent storage durations in cold room. The study was conducted at the Department of Animal Science, K.N.U.S.T Kumasi, Ghana, where a total of thirty eggs were obtained from the poultry section and sent to the Microbiology Laboratory for the determination of microbes. The samples analyzed included fifteen eggs each from egg-laying nest boxes and floor of a deep litter housing covered with litter. Eggs were then stored in a cold room at 16oC and 75% RH for up to 12 days. Microbial sampling over the eggshell surfaces was conducted at various points of collection (breeder house) and 4, 8, as well as 12 days following storage. The sampling of bacteria from each treatment was done using swab sticks, which were then dipped into separate test tubes containing Peptone water. Samples were cultured on Nutrient agar for total viable bacteria population and MacConkey agar for E. coli. Both media used to grow bacteria were sterilized by autoclaving at a temperature of 121°C for 20 minutes before culturing. The culture was incubated at 37°C for 24 hours and total microbial count performed on all colonies identified. Citrate and Tryptone were used for a biochemical test for the identification of E. coli on the incubated samples. A colony counter was used for enumerating bacteria colonies. Data were analyzed using the GLM procedure of SAS at P < 0.05. Prevalence evaluation of the microbes showed that eggs collected from the floor had a high bacteria load as compared to eggs laid in the nest. The general bacteria load and E. coli load on the egg samples reduced when stored in the cold room and almost reached zero at 12 days. The E. coli population was higher also higher in floor eggs versus nest eggs. For food safety and reasons of chick quality, it is important that hatching eggs are stored appropriately prior to incubation and this will reduce bacteria multiplication, reduce the practice of washing dirty eggs before incubation which can affect chicks and increase post hatch chick mortality.