The work must not be allowed to continue during the major Catholic event, which is expected to attract around 32 million visitors, according to a petition published on Tuesday by traders in the Borgo Pio neighbourhood near St. Peter's Square.
Having already written to Rome's mayor Roberto Gualtieri and the Vatican without success, hundreds of signatures have now been collected within a few hours.
More than 100 businesses with around 700 employees and an annual turnover in the tens of millions are affected.
It is "madness" that the Borgo Pio road, one of the most important traffic arteries in the neighbourhood of the same name, is to be renewed during the Holy Year celebrations.
The corresponding announcement from the city council was initially thought to be a mistake.
There were still a few million euros "left over" in the budget and "they absolutely have to be spent now", said the business people sarcastically.
Diggers, construction sites and diversions
The area has already been paralysed by huge construction sites for more than a year. Every day, people are subjected to a scenario of excavators, closed roads, dozens of diversions and police officers under constant stress.
At the same time, few workers can be seen on the construction sites, meaning that the measures are making little progress.
Business people fear that the private investments they made for the Holy Year will now come to nothing, jeopardising numerous jobs.
Pilgrims, for their part, would find building sites, dust and roadblocks instead of an appropriate welcome.
"We are making a final appeal to Mayor Gualtieri to ensure that the excavators stop and that our right to work quietly and effectively during the time of the greatest influx of visitors from all over the world is respected," they demanded.
The Holy Year is celebrated every 25 years, so there has been enough time for preparations.
If necessary, the road surface of Borgo Pio should be renewed after the Holy Year, but "not now!", the petition says.