His debut directorial venture, Musafir (1957), was not a success, but he persisted and received acclaim for his second film Anari in 1959. The film, crew and cast won five Filmfare Awards, with Mukherjee only losing the Best Director Award to his mentor, Bimal Roy.

In the following years he made numerous films. Some of his most notable films include: Anuradha (1960), Chhaya (1961), Asli-Naqli (1962), Anupama (1966), Aashirwad (1968), Satyakam (1969), Guddi (1971), Anand (1971), Bawarchi (1972), Abhimaan (1973), Namak Haraam (1973), Mili (1975), Chupke Chupke (1975), Alaap (1977), Gol Maal (1979), Khubsoorat (1980) and Bemisal (1982). He was the first to introduce Dharmendra in comedy roles, through Chupke Chupke, and gave Amitabh Bachchan his big break with Anand in 1970, along with Rajesh Khanna, he also introduced Jaya Bhaduri to Hindi cinema in his film Guddi. Having worked with his mentor, Bimal Roy as an editor, in films like Madhumati, he was much sought after as an editor as well.

Hrishikesh Mukherjee was honoured with the Dadasaheb Phalke Award by the Government of India, in 1999. Mukherjee was chairman of the Central Board of Film Certification and of the National Film Development Corporation. He was also awarded the Padma Vibhushan, the second highest civilian award for his contribution to Indian cinema by government of India in 2001 . The International Film Festival of India honoured him with a retrospective of his films in November 2005. He holds the distinction of working with almost all the top Indian stars since independence of India in 1947.

In later life, Hrishikesh Mukherjee suffered from chronic kidney failure and would go to Lilavati Hospital for dialysis. He was admitted to Lilavati Hospital in Mumbai early on Tuesday, 6 June 2006 after he complained of uneasiness. Mukherjee died few weeks later on 27 August 2006.