SPCA Otago inspector Sophia Tinirau said a motorist travelling past Taieri Lookout on Three Mile Hill on Thursday morning saw a man aiming a crossbow at a flock of roosters.
The flock was believed to be the one that appeared in the ODT on Tuesday.
SPCA Otago inspectors were deployed but could not find the alleged hunter or any arrows.
One of the flock’s roosters could not be found, but there was a pool of blood and black and white feathers at the lookout.
"We have concerns there was an act of ill-treatment towards the rooster and we would like to know who has done it," Ms Tinirau said.
SPCA feared other flocks in the city could be targeted.
Killing an animal in an inhumane manner was an offence under the Animal Welfare Act, she said.
Abandoning any animal was also an offence under the Act.
People with unwanted roosters should try to rehouse them or contact the SPCA for help.
"At the moment we have a few roosters we are trying to re-home — they make amazing companion animals and have all got individual personalities if you get to know them."
A police spokeswoman said police deployed an officer to the lookout on Thursday and searched the area but nobody was located.
On Monday, Dunedin City Council recreation planning and facilities manager Jendi Paterson said the council had deployed a pest contractor to assess the flock, including its potential ownership and whether it was creating a hazard.
A report from the contractor would dictate any future action taken by the council.
Yesterday, Ms Paterson said the contractor visited the lookout on Wednesday.
"The roosters were not causing a hazard so no action was needed."