The BBC One show will first introduce the "charmingly candid and utterly professional" consultant general surgeon Serena Campbell, played by Catherine Russell (The Inspector Lynley Mysteries, Pete versus Life).
Next to join the drama is "cocky and roguish" nurse and transplant coordinator Jonny Maconie (Michael Thomson), who "has no qualms about telling a few white lies to stir up some mischief".
Lastly, Dead Set and Campus star Chizzy Akudolu joins the cast as Jonny's "generous, plain-speaking and warm-hearted" best friend Mo Effanga, who joins Holby as registrar cardiothoracic surgery.
Holby City executive producer Johnathan Young said: "These are three very talented actors and I am delighted to welcome them to the cast.
"There are some dramatic and exciting storylines coming up on Holby City over the next few months and I know our new additions will have an immediate impact."
Holby City continues on BBC One and BBC One HD every Tuesday at 8pm.