A report published by the organisation said there were 116 deaths worldwide in 2014, including 29 in Brazil, 25 in Colombia and 15 in the Philippines.
Activists also faced abduction and other threats if they interfered in corporate or state interests, it added.
Last year saw a spike in killings related to hydropower programs.
Fourteen people died defending their land and rivers against dam projects.
Global Witness named Honduras as the most dangerous country for environmental activists, because of "regressive laws" and a climate of "near total impunity".
It has the highest number of killings per capita, with 111 deaths recorded since 2002.
Members of indigenous groups were increasingly involved in the "scramble for land and natural resources" and accounted for 40% of all deaths last year, it said.
In September four Peruvian tribal leaders were murdered on their way to a meeting to discuss ways to stop illegal logging.
Berta Caceres, an indigenous Lenca woman, told the report's authors that she had received numerous death threats because of her opposition to a dam that would force her community off their ancestral land.
She claims she has been forced to live a "fugitive existence".
Activists are often portrayed as enemies of the state, with some countries using anti-terror legislation to target them.
The campaign group urged governments and the international community to monitor, investigate and punish those behind what it called a hidden crisis.

The freighter sank after the collision on Wednesday night, about 11km (7 miles) off the Indonesian island Batam, Singapore officials said.
It was carrying 560 tonnes of fuel but there are no reports of an oil spill.
The Singapore Strait is one of the busiest shipping lanes in the world, although major accidents are rare.
Officials said the tanker Stolt Commitment, registered in the Cayman Islands, collided with freighter Thorco Cloud, which had the Antigua and Barbuda flag, around 20:00 local time (1200 GMT). The tanker had only minor damage.
The missing are among the freighter's crew of 12 men. The rest were rescued and are currently being treated in a Singapore hospital.
Anti-pollution craft have been put on standby in case of an oil spill, said Singapore's marine and port authority.
Killings of environmental activists have risen by 20% in the last year, according to campaign group Global Witness.
[NEXT_CONCEPT]
Authorities from Singapore and Indonesia are searching for six people, after a chemical tanker collided with a freighter in the Singapore Strait.