It's claimed Ofsted staff also spoke to children at Grindon Hall school in Sunderland about transsexuals, if they felt trapped in the wrong body and if tom boys were bullied.
It is a free school which means it receives state funding but is not under the local authorities control.
Head teacher Chris Gray has now formally complained to Ofsted about the inspection, which was carried out in November.
He claims girls aged 10 were asked what lesbians did after one child questioned how it was possible to have two mums.
Mr Gray said the inspection was "negative and hostile at every stage" and claims it was like the "data collected had to fit a predetermined outcome".
It's the latest in a long list of complaints made against Ofsted since new rules were introduced to ensure British values are being taught at schools.
Mr Gray said: "The view was that the Christian ethos of the school was adversely affecting our ability to be open non-discriminatory, and tolerant.
"I think that is upside down thinking."
He added: "In the feedback it was suggested that a response from one child to the effect of querying how it is possible to have two mums was viewed as indicating a lack of awareness of lesbian relationships.
"Actually, I understand the child concerned was merely thinking in biological terms.
"In addition, I have also heard reports of primary schoolchildren being asked if they knew of any boys or girls who thought they were in the 'wrong body'.
"Another parent has complained to me in writing that her 10-year-old daughter was asked if she knew what lesbians 'did'.
"Pupils were embarrassed and surprised to be asked questions about sexuality.
"I am also concerned that the manner and content of questioning of pupils crossed the line into harassment.
"I am further concerned that the questioning of pupils by Ofsted crossed a line into areas which fall outside its authority.
"Under the 'British values' requirements, our school is under a duty to promote respect and tolerance of those with different faiths and beliefs.
"However, I am alarmed that the questions asked of pupils sought to test the pupils' religious knowledge."
In a statement Ofsted said: "We are committed to making sure all pupils in England receive a broad and balanced education.
"One part of how we assess this is through talking to pupils to consider the extent to which they are being prepared for the next stages in their lives.
"Ofsted is not looking for answers to questions which are contrary to their faith. Nor do we require evidence that schools 'promote' other faiths. Instead, inspectors must ensure that pupils are able to express views which are neither intolerant nor discriminatory towards others.
"Ofsted takes all concerns about its work seriously. We will be considering the issues raised by the school as part of our normal quality assurance arrangements."
The regulator's been labelled 'barmy' by the Christian Institute.
Speaking on Premier's News Hour, spokesman Simon Calvert said:"There's clearly a problem at Ofsted. They are increasingly discredited. People say inspection teams can be very subjective.
"The Church of England has expressed concerns about the way Ofsted is going on these issues. The Roman Catholic Church has asked for an apology.
"The Government and Ofsted have got to do something about this because it's getting out of hand."
Listen to the full interview with Simon Calvert on Premier's News Hour with Des Busteed:
