Barksdale worked at the Department of Antibiotic Research of the Upjohn Company of Kalamazoo, Michigan (1943–52) and at the New York Botanical Garden.

While there, she discovered the chemical cycloheximide (trade name Actidione), an anti-fungal and anti-bacterial agent produced by the bacterium Streptomyces griseus.

In 1952, she married microbiologist Lane Barksdale and left the Upjohn Company, after which she and her new husband spent a year conducting research projects in Paris.

From 1955 to 1961, she expanded existing research on the species Achlya bisexualis, focusing on the fungus' unique sexual reproductive hormones.

It had been previously concluded by fellow mycologist, Dr.John Raper, that the development of sexual organs in A. bisexualis was mediated by the exchange of hormones between the male and female strains of the fungus.