Currently nurseries are required to have a designated safeguarding lead - but they do not necessarily have to be on the premises during opening hours.
The new guidance will mean an alternative member of staff will be required to take on the responsibility if the safeguarding lead is not present.
Purnima Tanuku, chief executive of National Day Nurseries Association (NDNA), said the guidance will require a "deputy" safeguarding lead to ensure someone is on the premises at all times.
"Having the deputy, or another senior member of staff who works opposite the manger, trained to be a deputy designated person for child protection, ensures appropriate cover at all times of the day," she said.
However, she said requiring two members of staff for safeguarding duties may be difficult for some nurseries.
"It could have implications for smaller settings which currently only have one designated lead and will need at least two from now on to ensure cover is available at all times," she said.
Ofsted has added a series of other measures to its guidance, including adding the welfare of children with special educational needs (SEN) and recognition of peer-on-peer abuse to safeguarding duties.
All staff and leaders are also now required to receive regular updates on safeguarding at least annually, and relevant training every two years, according to the guidance.
Tanuku said: "Safeguarding is everyone's business, so understanding current policy and ways in which children and young people can be abused is essential for staff and leaders to keep updated on current issues and effectively manage potential safeguarding situations and respond quickly to signs of potential abuse."