The RSPB says the lapwing, the curlew and the yellowhammer are all in decline
A conservation group wants an inquiry into why ministers have "failed to stop the loss of Welsh wildlife".
RSPB Cymru will hand in a 500-name petition to the assembly on Tuesday.
It said numerous species continue to decline, including birds such as curlew and yellow hammer, despite a target to halt biodiversity loss by 2010.
The assembly government said Wales was "not alone" but it was "absolutely committed" to halting the loss of wildlife and habitat.
Katie-Jo Luxton, RSPB Cymru director, said: "The decline of farmland birds, such as curlew, yellow hammer and lapwing has been particularly acute.
"The latest breeding birds survey shows the numbers of yellow hammer in Wales has decreased by nearly 40% since 1995.
Public money is also being wasted on schemes that have a negative impact on the environment...
Katie-Jo Luxton, RSPB Cymru director
"While a recent species-specific survey has estimated the number of curlew has declined by 81% since 1996."
She said the assembly government had made "some progress" to protect biodiversity, describing the new Glastir agriculture-environment scheme as "urgently needed".
"However, much more is needed as many policies and projects fail to fully address their environmental impacts," said Ms Luxton.
"Public money is also being wasted on schemes that have a negative impact on the environment, such as the subsidy given to the intra-Wales airline."
RSPB Cymru said the assembly government had failed to reach its target because of a lack of resources and political ownership.
The charity said extra investment to ensure the recovery of biodiversity was under way by 2026 was unlikely without the evidence that an inquiry would provide.
Ms Luxton added: "Against the backdrop of the current economic situation, it might seem the wrong time to invest in our wildlife and natural environment.
"But with an estimated £6bn of our gross domestic product being dependent on the environment in Wales, can we really afford not to invest?"
One of the impacts of climate change has been the departure of species from their previous habitats
Environment Minister Jane Davidson
The assembly government said it was "absolutely committed to halting the loss of wildlife and habitat" and had issued a written statement to provide an update on its progress and plans for 2010, the international year of biodiversity.
It also said that Wales was "not alone" and so far no European country had met the 2010 target of halting loss for every species and habitats.
Environment Minister Jane Davidson will be hosting a conference on biodiversity which aims to bring together ideas and best practice, and will involve specialists including RSPB Cymru.
She said: "The assembly government has already gone the extra mile to make sure that public authorities understand that halting the loss of species and habitats is part of their Natural Environment and Rural Communities (NERC) biodiversity duty.
"No other government has done that.
"One of the impacts of climate change has been the departure of species from their previous habitats. I am aware of this and take the issue very seriously."
She said while the assembly government had not halted the overall loss, there were a number of improvements for key species and habitats.
These included arable field margins, the Deptford pink wildflower, the sand lizard, and the lesser horseshoe bat.
The call for an inquiry comes as the RSPB launches what it calls its biggest ever campaign, Letter To The Future.
The campaign urges politicians to consider the health of the planet for future generations when making decisions about where to make cuts and where to invest.
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