The microbiological and storage quality of expressed human breast milk was studied between July and December, 2016. One hundred and twenty working class lactating mothers and thirty lactating mothers visiting Imo State University teaching Hospital Orlu for various health challenges were recruited for the study. They were requested to express 60ml of their breast milk into sterile containers. The milk sample collected from each mother was distributed 10ml into each of 3 sterile containers. One set was heated at 100OC for 1hour in a water bath, 1 set was stored in a refrigerator at – 4OC for 5 days and 1 set was stored on the bench at ambient temperature without any treatment. 0.1ml of each sample was inoculated on laboratory culture media before commencement of storage and 2hours, 6hours, 12 hours, 24 hours and 5days post storage. Eight genera of bacteria: Stapylococcus aureus, Streptococcus viridians, Diphtheroides, Escherichia coli, Klebsiella species, Lactobacillus species, Pseudomonas species and Salmonella species, were isolated from expressed human breast milk samples. The most prevalent bacterium in the milk samples was Staphylococcus epidernidis, followed by Escherichia coli. The least prevalent bacteria were Pseudomonas aeruginosa, Salmonella species and Diphtheroides. No bacterium was isolated from milk samples heated at 100OC and stored in a refrigerator. The total heterotrophic bacterial counts of the milk samples collected from healthy working mothers ranged from 3.2 x 103 to 8.2 x 103cfu/ml, while that of health challenged mothers ranged from 4.3 x 103 to 1.6 x 104cfu/ml. As shown, the bacterial counts of the samples. Out of 30 samples collected from health – challenged mothers, 9 (30%) had total heterotrophic bacteria count ranging from 1.2 x 104 to 1.6 x 104cfu/ml, 21 (70%) had total bacteria count ranging from 4.3 x 103 to 8.6 x 103cfu/ml. Analysis of the data using chi square showed significant difference (p < 0.05) in the total heterotrophic bacterial count of breast milk between healthy working mothers and health challenged mother.