Sir Lindsay Hoyle told the chamber he was “very worried” after a “large number” people were contacted to isolate.
Unions representing staff based in Parliament have previously written to the Speaker to push him to reconsider guidance for post-July 19, in which politicians will be encouraged to wear a mask but will not be mandated to have one on.
It is compulsory, however, for staff on the estate to continue to have to wear a covering.
In reply to the unions, Sir Lindsay last week said there was “no meaningful way” for him to force MPs to wear a face covering from Monday.
Sir Lindsay, making a statement at the start of proceedings on Monday, said: “With the increasing infection levels in the community and people having to isolate, I strongly advise that everyone on the estate should wear face coverings when moving around, assessing catering venues and in the areas they consider crowded, including the chamber.
“We have four sitting days before the House rises.
“I really want us to behave safely, responsibly, during these few days.
“None of us wants to risk taking Covid back to our families, staff or constituents and I’m sure we will want everyone working on the estate to feel safe and secure, and to ensure the business of this House continues.
“I’ve got to say we’ve certainly got a large number of people that have been contacted so I’m very worried about the outcome.
“Four days to go, I want us all to have a good break and enjoy our holidays so hopefully we will take it seriously and not push the limits for the sake of it.”
Most MPs in the sparsely attended chamber were wearing masks at the start of Ministry of Housing Communities and Local Government questions.