Holt's first film role came in 1950, acting as Octavius Caesar in Julius Caesar. His career as a voice artist began with the 1968 short film Johnny Learns His Manners, for which he provided all of the voices. He later appeared in such works as Bedknobs and Broomsticks, several animated television specials with Dr. Seuss, for example, The Lorax (1972), Dr. Seuss on the Loose (1973), The Hoober-Bloob Highway (1975) and The Grinch Grinches the Cat in the Hat (1982) and the animated film version of Charlotte's Web as Homer Zuckerman.

Holt appeared in a variety of different works, including animated films for both adults (the 1974 sequel The Nine Lives of Fritz the Cat) and for children, as well as voice and acting work in live-action films (for the blaxploitation film Abby, Holt provided the voice of the Demon). In 1975, he was the voice of Grape Ape on The New Tom and Jerry Show. The same year, Holt provided the voice of Avatar in Ralph Bakshi's film Wizards. Avatar's voice was an imitation of actor Peter Falk.

In 1982, Bob Holt played the title role in Marvel Productions' animated The Incredible Hulk (1982 TV series), also in the process creating a library of stock roars that would be used for many years afterwards.