Fish of all sizes were spotted swimming erratically and then floating to the surface in the stream that runs through the coastal village last week, and residents are worried that pollution may be to blame.
Grant Course, a marine scientist who lives in Powfoot, said he is concerned that a whole generation of fish may have been wiped out.
He said: “This happened two years ago and it took until this year for the trout to return.
“If you look at the dead flatfish, these are the baby founders that hatched out in spring and their nursery areas are streams like this.
“They have all been killed, the trout from here to Cummertrees have been killed, the visiting Detroit that spawn here have been killed along with any eggs and fry, same with any salmon.
“We had otters feeding here and they have lost a food source, same with the kingfishers, so they will leave.
“The insect life will have suffered too.”
Other residents who had been walking near the Pow Burn last weekend said they thought the water looked very dirty, and one woman said her dog had been sick after paddling in the stream.
A similar incident occurred in the Pow Burn back in August 2018, where hundreds of dead fish were discovered.
On that occasion an officer from the Scottish Environment Protection Agency (SEPA) collected samples as part of an investigation into possible sources of pollution, although the results of the investigation were never made public.
A spokesman for SEPA yesterday said: “Every day SEPA works to protect and enhance Scotland’s environment and takes all reports of pollution seriously.
“SEPA was informed of a number of dead fish found in the Pow Burn in Powfoot on Wednesday June 25 and is currently investigating to establish the cause of the incident.
“Pollution incidents can be reported via our website at www.sepa.org.uk/report.”