Henley Villa, a housewares and gifts shop which opened in Duke Street more than 40 years ago, is one of two stores to have closed this week.
Clothes shop Dashwood Boat Club in Hart Street has shut after only six months.
Mike Noades founded Henley Villa in 1971 and retired in 2000 when his son David took over.
Mr Noades jnr says his decision to close the shop was down to rising costs and loss of custom.
He said: “We have found business getting tougher and tougher as people have become more cautious about what they spend their money on.
“It is tough with the overheads, which keep on rising, and I felt it was time to close.
“There have always been issues with parking in the town but in the past two years trade has suddenly turned. Footfall in the town has definitely dropped.”
Mr Noades, who is married with five children and lives in Earley, said he had lost customers to the internet.
“We feel we have become more of a showroom,” he said. “We are still getting people in the shop but they are just looking rather than buying.
“I also started to notice that a lot of my suppliers deal directly on the internet.
“That is the way things are going and I have had the foresight to see that. I am quitting while I am ahead. My dad quite agrees with what I am doing — he says it is the best decision in the circumstances — but it is not a decision you like to make.”
The shop was selling all its stock at half price before closing for the last time.
Mr Noades, a trained decorator, said he had enjoyed running the shop.
He said: “One day my dad needed some help and I ended up staying for good.
“We have seen lots of shops come and go and empty units fill up again. Henley has the pull because it is a nice town to come to. So many people have said that it is a shame — people who have known the shop for many years and are sad to see it going.
“I would like to thank our customers for all their support over the years. We have met some great characters.”
The Dashwood clothing brand launched its first store in March with Olympic gold medallist Alex Gregory and Team GB rower Vicky Thornley as ambassadors. The shop’s interior was designed to look like a traditional boat club. It included a gym-themed room which stocked casual basics as well as a “club house”, which was a formal collection.
At the time of the opeing, founder and managing director Richard Cross said: “Our first shop had to be in Henley because it’s the home of rowing.”
He has refused to comment on the shop’s closure, saying it was “sensitive”.
It is the second clothes store to close since July following the demise of Last Man Standing, also in Hart Street. Eight shops in the town have now closed since early June.
Town centre manager Peter McConnell said: “Henley is an incredibly tricky trading environment. Everything that Henley Villa sells you can buy in John Lewis and on the internet.
“It is a shame the shop is closing down because it was an institution and in a very good position. This reflects how the nature of shopping has changed. The internet is the scourge of the modern shop, especially in small towns like Henley.”