Mary Engle Pennington was born in Nashville, Tennessee, to Henry and Sarah (Malony) Pennington. She was raised in a Quaker family, which instilled in her a strong sense of independence and a commitment to social responsibility.

Pennington displayed an early interest in science, particularly chemistry. This was unusual for girls at the time, but her parents encouraged her intellectual pursuits.

Pennington audited classes in chemistry at the University of Pennsylvania, since the university did not formally admit women to its chemistry program.

Pennington completed the requirements for a Bachelor of Science degree in Chemistry, Biology, and Botany from the University of Pennsylvania, but did not receive it. It was only much later in 1905 that she was formally awarded the degree.

Pennington earned her Ph.D. in Chemistry from the University of Pennsylvania, focusing on physiological chemistry. Her dissertation explored derivatives of columbium and tantalum.

Pennington began teaching as a lecturer at the University of Pennsylvania, in the field of botany.

She worked as the Director of the Clinical Laboratory at the Women's Medical College of Pennsylvania. She conducted research and lectured on hygiene and related fields, expanding her knowledge of bacteriology.

Pennington began her career in public service as a bacteriologist at the Bureau of Chemistry, U.S. Department of Agriculture, in Philadelphia. This marked the beginning of her work in food safety and preservation.