More than 1,000 people gathered in Piazza del Popolo in central Rome shouting "No Green Pass!" and "Freedom!".
Thousands more marched in Milan, with some wearing Star of David badges, like those worn by Jews in Nazi-era Germany, with the words "not vaccinated", the ANSA news agency reported.
Around 100 people from the "No Vax" movement also gathered in Naples, objecting in particular to vaccinations for children, shouting "Hands off the children" and "Shame! Shame!".
The Green Pass, an extension of the EU's digital Covid certificate, became compulsory in Italy on Friday to enter cinemas, museums and indoor sports venues or to eat indoors at restaurants.
It proves bearers have either been vaccinated with at least one dose, have recovered from Covid-19 within the past six months, or have tested negative in the previous 48 hours.
School and university staff will need the pass, as will university students, while from September 1 it will be required on domestic flights and long-distance trains.
It is the latest tool used to tackle the coronavirus pandemic, which has left more than 128,000 people dead in Italy and last year brought the economy to a shuddering halt.
A successful vaccination campaign has helped turn the tide in recent months, with more than 63 percent of the population over the age of 12 now fully jabbed.
However, the spread of the highly contagious Delta variant is causing concern, with almost 7,000 new cases reported on Saturday.
