Investigation into the effect of storage period and temperature on the shelf life of Human Breast Milk (HBM) was carried out to assess the quality and fitness of expressed HBM after storage for 3 and 6 hours at 4°C and 26°C. The control sample was collected from a nursing mother using standard hygienic method while other samples were collected aseptically from nursing mothers, day care centers, crèches and various homes. The samples were conveyed to the laboratory for microbial analyses. There was no fungal growth observed in any of the samples. The mean bacterial count obtained for the fresh HBM was 1.53 × 10³ and 1.85 x 10³ Cfu/mL at 4°C and 26°C respectively after 3 hours of storage. At 4°C the bacterial load was 1.61 x 10³ Cfu/mL and 2.16 x 103 at 26°C after 6 hours. Before and after 3 hours of the storage at 4°C, the control HBM had no microbial load, but has a value of 0.4 × 10³ Cfu/mL at 26°C after 6 hours. The bacteria isolated from milk samples after storage are Bacillus subtilis, Corynebacterium spp., Lactobacillus salivarus, Staphylococcus aureus, Staph. epidermidis, Lactobacillus acidophilus, Neisseria catarrhalis and Streptococcus lactis. It is recommended that the length of storage should not exceed 6 hours at 26°C and 24 hours at 4°C.