Susanne Wilkinson said she was trying to uphold her beliefs about marriage at the Swiss Bed and Breakfast in Cookham, Berkshire.
At a court hearing this week, the judge accepted the sincerity of her Christian beliefs and that she had also refused to allow unmarried heterosexual couples from sharing a double bed.
However, the judge ruled that the policy broke equality laws by discriminating against Michael Black and John Morgan.
She was ordered to pay damages on the grounds of hurt feelings to the couple.
Mrs Wilkinson is considering appealing the ruling.
She said: “Naturally, my husband and I are disappointed to have lost the case and to have been ordered to pay £3,600 in damages for injury to feelings. We have the option to appeal, and we will give that serious consideration.
“We believe a person should be free to act upon their sincere beliefs about marriage under their own roof without living in fear of the law. Equality laws have gone too far when they start to intrude into a family home.
“People’s beliefs about marriage are coming under increasing attack, and I am concerned about people’s freedom to speak and act upon these beliefs. I am a Christian, not just on a Sunday in church, but in every area of my life – as Jesus expects from his followers.
“That’s all I was trying to do and I think it’s quite wrong to punish me for that, especially after enduring over two years of vile abuse and threats. We find this a strange justice in a society that aspires to be increasingly tolerant.”
Mrs Wilkinson’s legal defence was paid for by The Christian Institute, which is working to protect the civil liberty of Christians.
Spokesman Mike Judge said: “Yes, Mrs Wilkinson’s B&B is a business, but it’s also a family home.
"The law should be more flexible in allowing people to live according to their own values under the own roof.
"A bit more balance is needed, rather than allowing one set of rights to automatically suppress another.”