O'Byrne had served in the United States Army before becoming an elementary school teacher. He later moved to New York City to pursue acting. O'Byrne successfully entered the profession after starring in the Broadway production of Duel of Angels opposite actress Vivien Leigh in the late 1950s. O'Byrne studied acting under Stella Adler and dance with Martha Graham. He resided in the same New Yorkapartment building as actors James Farentino and Marlon Brando, befriending both of them early in their careers. O'Byrne soon moved to Los Angeles to pursue opportunities in film and television. O'Byrne credits during his career included numerous film and television roles. The Hollywood Reporter described many of his characters as "quiet,." Additionally, he appeared in more than 200 television commercials. He was particular active within the television industry during the 1960s and 1970s. In 1962 O'Byrne appeared as Ned Carlin on the TV western The Virginian in the episode titled "The Accomplice." He appeared in six episodes of Get Smart as Hodgkins, the assistant to the Chief. O'Byrne played Robin's high school principal in the 1960s Batman television series. He regularly appeared as a mortician in The Munsters, as well as the series Occasional Wife, in which he played the Man-in-Middle, and as Mr. Beasley, the mailman, in Blondie. He made a guest appearance on Perry Mason in 1965 as murder victim Horace Lehigh in "The Case of the Baffling Bug." His other lengthy television credits included guest appearances on Alfred Hitchcock Presents, Murder, She Wrote, The Bob Newhart Show, Happy Days, The Bill Cosby Show, The Partridge Family, Sanford and Son and Gunsmoke. O'Byrne film credits included Spaceballs, Gunfight in Abilene, Marnie, The Shakiest Gun in the West, The Million Dollar Duck, Gus, Love at First Bite, The Apple Dumpling Gang Rides Again and Zapped!.
O'Byrne also worked as an acting teacher and coach to many early actors. Much of his instruction emphasized repetition and scene study. Film studios sometimes recommended that younger actors work with O'Byrne to improve their performances. O'Byrne coached and mentored many then-unknown film and television actors. Forest Whitaker, Jimmy Smits and Lou Diamond Phillips visited his acting classes early in their careers. He also coached acting to Pam Dawber, Christopher McDonald, Bonnie Bedelia, Bill Allen and Marj Dusay. In particular, O'Byrne is credited with launching the acting career of Nick Nolte. O'Byrne, who often coached college students, was working with Nolte's college roommate at the time. O'Byrne asked Nolte, who was not an actor at the time, to read a scene they were working on. O'Byrne reportedly recognized Nolte's talent and began coaching him as well. Nolte spent nearly a year sleeping on O'Byrne's couch while working with him to become an actor. O'Byrne cast Nolte in his production of The Last Pad, by playwright William Inge, which effectively launched Nolte's professional career. Professionally, O'Bryne was a member of the Actors' Equity Association, the American Federation of Television and Radio Artists and the Screen Actors Guild. He served on the Emmy Nominating Committee, based in Los Angeles.
O'Byrne retired from acting in the 1990s to care for his sister, Henrietta Bouyea, who was in failing health. He moved to Pacifica, California, after retiring, where he lived for the last sixteen years of his life. O'Byrne died on December 4, 2009, in Pacifica, California, at the age of 78. His funeral was held at the Church of the Good Shepherd in Pacifica. Actor Christopher McDonald, a close friend and former student, read the eulogy at O'Byrne's funeral.
