Benjamin Walden Thomas was born on August 13, 1821, in Genesee County, New York.

He was a lumber merchant and microscopist. Around 1841 he moved to Chicago, where he initially worked as a shop clerk. In 1843, he took a similar position with a lumber business, which he purchased in 1844 and then expanded over the next three decades. In 1871, Thomas sold his business and devoted a considerable amount of time to microscopic investigations of life in his area of North America, with a particular focus on diatoms and freshwater sponges. He joined two local scientific societies, the Chicago Academy of Sciences, and the Illinois State Microscopical Society (ISMS). Thomas served as President of the ISMS in 1881. In 1882, he joined The American Society of Microscopists and later served as its officer. He was elected Fellow of the Royal Microscopical Society in 1884.

In 1848, he married Ann Augusta Wilcox (1829–1910). He died on January 1, 1905, in Chicago, Illinois.