Nachtwey started working as a newspaper photographer in 1976 at the Albuquerque Journal. In 1980, he moved to New York and began working as a freelance photographer. In 1981, Nachtwey covered his first overseas assignment in Northern Ireland illustrating civil strife. He has documented a variety of armed conflicts and social issues, spending time in South Africa, Latin America, the Middle East, Russia, Eastern Europe, the former Soviet Union shooting pictures of war, conflict and famine, and images of socio-political issues (pollution, crime and punishment) in Western Europe and the United States. He currently lives in New York City.

In 1994, Nachtwey was covering the upcoming elections in South Africa, the first non-racial ones in decades. As an associate of the Bang-Bang Club, he was at the scene when Ken Oosterbroek was killed and Greg Marinovich was seriously injured.

Nachtwey had been injured previously in his work, but it was during his extensive coverage of the United States invasion of Iraq that he received his first combat injury. As Nachtwey, along with TIME correspondent Michael Weisskopf rode in the back of a humvee with the United States Army "Tomb Raiders" Survey Platoon, an insurgent threw a grenade into the vehicle. Weisskopf grabbed the grenade to throw it out of the humvee, but it exploded in his hand. Two soldiers were injured in the explosion, along with the TIME journalists. Nachtwey managed to take several photographs of medic Billie Grimes treating Weisskopf before passing out. Both journalists were airlifted to Germany and later to hospitals in the United States. Nachtwey recovered sufficiently to return overseas to cover the tsunami in Southeast Asia of December 26, 2004.

Nachtwey has worked with TIME as a contract photographer since 1984. He worked for Black Star from 1980 until 1985 and was a member of Magnum Photos from 1986 until 2001. In 2001, he was a founding member of the VII Photo Agency.

Nachtwey was present during the September 11, 2001 attacks on the World Trade Center, and produced a well known related body of work. He also compiled a photo essay on the effects of the Sudan conflict on civilians.

Nachtwey photographs have been exhibited throughout Europe and the United States and he has received numerous prizes and awards including the World Press Photo award in 1994. Nachtwey has also been awarded the Overseas Press Club's Robert Capa Gold Medal in 1983, 1984, 1986, 1994 and 1998. In 2001, the documentary War Photographer was released, focusing on Nachtwey and his work. Directed by Christian Frei, the film received an Academy Award nomination for best documentary film.

Nachtwey received the prestigious Dan David Prize in 2002 for his haunting photos aimed to burden viewers with an uncomfortable awareness that will force them to seek justice and change.

In 2006, Nachtwey was awarded the 12th Annual Heinz Award in Arts and Humanities from the Heinz Family Foundation for his body of work, an honor that includes a monetary prize of US $250,000. Nachtwey is one of three winners of the 2007 TED Prize. Each recipient was granted $100,000 and one "world-changing wish" to be revealed at the 2007 TED conference, in Monterey, California. Many members of the TED Community, and a group of world-class companies, have pledged support to help fulfill the wishes. Nachtwey's wish, revealed March 8, 2007, is this: "There's a vital story that needs to be told, and I wish for TED to help me gain access to it and then to help me come up with innovative and exciting ways to use news photography in the digital era." Those who wish to help him will sign an NDA and help him "gain access to a place in the world where a critical situation is occurring and fully document it with photography; set a date to unveil the pictures and find a series of innovative ways to create powerful impact with them, using novel display technologies and the power of the Internet as well as media; and use the campaign to generate resources for organizations that are working to address and transform the situation." Early results of this work have been unveiled at XDRTB.org to document extensively drug-resistant tuberculosis throughout the world.

In 2008, Nachtwey exhibited a series of original photographs at Le Laboratoire in Paris, France. The exhibit entitled "Struggle For Life" documented the human toll of TB and AIDS presented the work of Nachtwey with text by Dr. Anne Goldfeld of work they began together in Cambodia in 2003 as well as photos from Thailand, Africa and Siberia. The work was accompanied by film portraits of Nachtwey and several leading medical scientists participating in the Attention! Symposium by American filmmaker Asa Mader.