In June, the trunks of two pines were drilled up to 15 times in West Cliff Green, Bournemouth, Dorset, close to the former St George's Hotel site.
A third pine was damaged the following month and a "crystal-like" substance was discovered on its bark.
Tests showed the substance was the herbicide glyphosate, the borough council said.
Nick Colledge, from Bournemouth Borough Council, said the samples from all three trees "tested positive for very high levels of the herbicide glyphosate".
"This would seem to remove any doubt that the holes were drilled with the sole purpose of administering a toxin with the intention of destroying the trees," he said.
The authority now hopes to install CCTV cameras in the area in a bid to prevent further tree attacks.
Dorset Police is investigating the damage, but no arrests have been made.
Traces of poison have been found in trees that were damaged in a seaside resort, tests have revealed.