Conservationists are pressing for tougher laws to combat wildlife crime on Scottish grouse moors after a record number of poisoning cases against birds of prey such as golden eagles and red kites last year.

The RSPB Scotland said ministers should make grouse moor owners legally responsible for attacks on birds of prey on their estates because existing laws and voluntary codes had failed.

The conservation charity said the latest official figures showed there were 46 proven poisoning incidents targeting birds of prey last year – the highest total in two decades – and another seven cases where illegal poisons were found on shooting estates.

The cases included the deaths of two rare golden eagles, four red kites, 21 buzzards and a sea eagle. As well as poisoned baits such as rabbits and eggs, the RSPB included cases where cats, magpies and ravens were poisoned accidentally by pesticides they believe were laid to kill birds of prey.

Some gamekeepers on grouse moors and pheasant shooting estates target birds of prey because they eat game birds kept for commercial and private shooting.

The record level of incidents, which compares to an average of 27 cases a year since 1999, is a significant setback to efforts by the Scottish government to tackle wildlife crime and an embarrassment to Highland estate owners who insist they abhor such attacks .

Over the last three months 255 Scottish estates, including prominent landowners such as the Earl of Hopetoun and Mohamed Al Fayed, have signed an unprecedented statement condemning offences against birds of prey.

Duncan Orr-Ewing, head of species for RSPB Scotland, said the current legal and voluntary approaches were not working and prosecutions of gamekeepers were very rare. In addition to the 46 poisoning cases, there were nine confirmed incidents involving traps, shooting and nest disturbance, and 19 classed as "probable".

He urged Roseanna Cunningham MSP, the environment minister in Edinburgh, to implement a recommendation from the HM Inspectorate of Constabulary and Crown Office in 2008 to investigate making landowners legally responsible for crimes by their gamekeepers with "vicarious liability" legislation.

Similar laws mean that pub landlords are liable for underage drinking on their premises and company owners are liable for health and safety breaches. But the recommendation has not been included in a new wildlife and countryside bill now going through the Scottish parliament. Other sanctions could include tougher licensing, said Orr-Ewing.

He said that voluntary agreements with landowners had failed. "There is going to be a need for stronger sanctions," he said. "We have to get the right laws in place to tackle this; they've had 20 years to sort this out voluntarily and nothing has improved."

A Scottish government spokeswoman said ministers believed there already was strong legislation in force to tackle this "unacceptable" type of crime, but she added ministers would give "careful consideration" to any proposals from MSPs to strengthen the law in the new bill.

In a joint statement, Scotland's two major landowners' organisations, the Scottish Rural Property and Business Association and the Scottish Estates Business Group, said their opposition to wildlife crime was "absolutely clear", but said there was no need for further legislation.

"There is ample legislation in existence to deal with this problem and we support the full weight of the law being brought to bear on those using illegal poisons," it said.

The RSPB's claim of a record year was "misleading", it added. A Scottish government report earlier this year, which uses a different method of counting cases and does not include other species or the discovery of poisons, showed the actual number of dead poisoned birds recovered had remained constant for the last three years, except in 2008, when a large number of birds were found.