He was born in Winfield, Kansas, the son of william Baird Pallette (1858–?) and Elnora "Ella" Jackson (1860–1906). His sister was Beulah L. Pallette (1880–1968).

Pallette began his silent film career as an extra in about 1911. His first credited appearance was in the one-reel short western/drama The Fugitive (1913) which was directed by Wallace Reid for Flying "A" Studios at Santa Barbara.

When the "blow-up" he anticipated failed to materialize after two years, he began disposing of the Oregon ranch and returned to Los Angeles and his movie colony friends but, after working steadily from 1913 to 1946, never appeared in another movie.

Quickly advancing to featured status, Pallette appeared in many westerns. He worked with D. W. Griffith on such films as The Birth of a Nation (1915) and Intolerance (1916). At this time, he had a slim, athletic figure, a far cry from his portly build later in his career. He became famous, starring as the slender sword-fighting swashbuckler Aramis in Douglas Fairbanks' 1921 version of The Three Musketeers, one of the great smash hits of the silent era.

After gaining a great deal of weight, he became one of the screen's most recognizable character actors. In 1927, he signed as a regular for Hal Roach Studios and was a reliable comic foil in several early Laurel and Hardy movies. In later years, Pallette's weight may have topped out at more than 300 pounds (136 kg).

The advent of the talkies proved to be the second major career boost for Pallette. His inimitable rasping gravel voice (described as "half an octave below anyone else in the cast") made him one of Hollywood's most sought-after character actors in the 1930s and 1940s.

After an early career as a slender leading man, Pallette became a stout character actor. He had a deep, gravelly voice, and is probably best-remembered for comic character roles such as Alexander Bullock, Carole Lombard's character's father, in My Man Godfrey (1936), as Friar Tuck in The Adventures of Robin Hood (1938) starring Errol Flynn, and his similar role as Fray Felipe in The Mark of Zorro (1940) starring Tyrone Power.

Pallette was cast as the father of lead Actress Jeanne Crain in Army Wives (released in 1944 as In the Meantime, Darling). Director Otto Preminger clashed with Pallette and claimed he was "an admirer of Hitler and convinced that Germany would win the war". Pallette also refused to sit down at the same table with black actor Clarence Muse in a scene set in a kitchen. "You're out of your mind, I won't sit next to a nigger", Pallette hissed at Preminger. Preminger furiously informed Fox studio head Darryl F. Zanuck, who fired Pallette. Although Pallette remains in scenes he already had filmed, the remainder of his role not yet shot was eliminated from the script.

In 1946, convinced that there was going to be a "world blow-up" by atom bombs, the hawkish Pallette received considerable publicity when he set up a "mountain fortress" on a 3,500-acre (14 km) ranch near Imnaha, Oregon, as a hideaway from universal catastrophe. The "fortress" reportedly was stocked with a sizable herd of prize cattle, enormous supplies of food, and had its own canning plant and lumber mill.

Eugene Pallette died at age 65 in 1954 from throat cancer at his apartment, 10835 Wilshire Boulevard, in Los Angeles. His wife, Marjorie, and his sister, Beulah Phelps, were at his side. Private funeral services were conducted on Saturday, September 4, 1954, at the Armstrong Family Mortuary. His cremated remains are interred in an unmarked grave behind the monument of his parents at Green Lawn Cemetery in Grenola, Kansas. He has a star on the Hollywood Walk of Fame at 6702 Hollywood Boulevard for his contribution to motion pictures.