In an open letter, the students - who are aged between 13 and 15 - called on Rishi Sunak to tackle the issue head on when the Government issues its long-awaited transgender guidance to schools.
The girls - who used only their first names to sign their letter for fear of a backlash from trans activists - said single-sex facilities were an 'essential safeguarding feature' of schools.
But they warned that many schools had started introducing mixed toilets without consultation to appease transgender and Left-wing activists.
The five pupils, who identified themselves as Cynthia, Marilyn, Sonja, Olivia and Ellie, said many female pupils found mixed-sex toilets 'intimidating, humiliating and downright dangerous'.
They called on Mr Sunak, who is a father of two daughters, to uphold their rights.
'There is no justifiable reason to introduce dangerous and uncomfortable spaces into schools, and the Government has a duty to ensure that toilets in schools are safe and dignified,' they wrote.
The letter warns that in 'many schools, girls are so uncomfortable with having to use the toilets there that they simply don't go at school, risking a urinary tract infection'. They decided to write to Mr Sunak after almost 12,000 people signed an online petition launched by one of the girls calling for single-sex spaces to be protected in schools.
The letter cites a school in Southampton where there were recent protests against the introduction of 'gender-neutral' toilets amid claims that boys were photographing girls in the gaps above or below toilet cubicles.
A teenage boy was arrested after police investigated reports that a number of girls were being sexually assaulted in mixed-sex toilets at another school in Essex.
The girls said being forced to share toilets with boys was unpleasant for female students who were menstruating, adding: 'Girls must have private spaces where we can sort this out with dignity, away from potential shaming from boys and the humiliation of having everyone know you're on your period.'
Cynthia, 15, said her secondary school had as many mixed-sex toilets as single-sex ones. She told The Mail on Sunday: 'The mixed-sex toilets are so disgusting and unhygienic. Very few people use them apart from the boys, so really they're just another set of boys' toilets.
'You get boys graffiti-ing images of male genitalia on the wall, there's always a load of pee on the floor and even on the wall sometimes.
'But if I'm in a class such as English, for instance, there are now no girls' toilets nearby, so I have to go out of the building and walk some distance to a whole other block in the school to find some.'
Marilyn, 14, who attends a school of more than 1,000 pupils with just one set of facilities each for girls and boys, and one mixed one, said: 'Girls definitely feel unsafe. Being around boys from older years is generally very intimidating, so there would definitely be a fear factor being with them in the toilets.'
The letter to Mr Sunak comes as increasing numbers of schools are introducing mixed-sex toilets, often on the advice of trans groups who claim it is a breach of equality laws for trans pupils to be prevented from accessing facilities of the gender they identify as.
But the Government appears to be pushing back against this trend. This month it announced changes to regulations, insisting all new buildings must provide separate single-sex toilets for men and women. But with less than two weeks to go until children return to the classroom, it remains unclear whether the Government's transgender guidance for teachers will clarify the issues over gender-neutral bathrooms in schools.
Last night, Labour MP Rosie Duffield urged the Government to publish the guidance, adding: 'I hear from school pupils and students who are really uncomfortable with this. They are simply asking for their right to privacy and dignity to be respected with the provision of single-sex spaces.'
A Department for Education spokesperson said:
"We have been clear that schools must continue to meet their existing legal duties to protect single sex spaces.
"The Education Secretary is working closely with the Minister for Women and Equalities to publish guidance for schools at the earliest opportunity."
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