Paramedics and firefighters were called to the falls entrance near Wilson Street East in Ancaster around 3 p.m.
Hamilton fire spokesperson Claudio Mostacci said fire crews came to assist, but the woman was pronounced dead at the scene by paramedics.
First responders say the branch likely fell about 40 feet, and that the woman was "obviously dead" when they arrived.
The Hamilton Conservation Authority issued a statement and said the Tiffany Falls Conservation area is closed until further notice.
"Staff were shocked and saddened to learn of the death of a female hiker at Tiffany Falls Conservation Area," the statement reads.
"We would like to extend our deepest sympathies to her family and friends at this time. Tiffany Falls is currently closed while we are looking into the causes of the incident."
Burlington resident Corina Ciobanu tweeted about the incident not long after it happened to express her shock:
Hamilton police spokesperson Const. Steve Welton told CBC Hamilton that he couldn't comment on the incident as it is a coroner's case.
Regional coroner Dr. Jack Stanborough couldn't immediately be reached for comment.