The former Tory MP made the reference to Lib Dem AM Kirsty Williams and Plaid Cymru leader Leanne Wood in his maiden Senedd speech.
But Ms Williams accused Mr Hamilton of using "blatant sexist language".
Presiding officer Elin Jones called on AMs to be respectful.
Ms Wood and Ms Williams backed Mr Jones in being reinstated as first minister, which was confirmed during the session.
During Mr Hamilton's speech the UKIP AM said he regretted the role Plaid Cymru had played and added Ms Williams had "managed to prop up this tottering administration".
He added: "So I'm afraid that these two ladies have just made themselves political concubines in Carwyn's hareem. What a gruesome prospect that must be."
He asked what the two had had in reward for "the sacrifice of their political virtue".
Mr Hamilton later, reflecting on the deal between Labour and Plaid, also accused Ms Wood of being a "very cheap date indeed".
He also accused the first minister being "wholly impotent" over the steel industry because of his commitment to the EU, "which I suppose the members of his harem may be rather satisfied with".
Speaking after the debate, he said he did not regret making the remarks.
He told BBC Wales: "Je ne regrette rien".
But Ms Wood said: "This sexist language has no place in the National Assembly for Wales, or in society, and we will not stand for sexism, homophobia or racism."
Following the comments Elin Jones, presiding officer, has warned that members must respect each other during Senedd debates.
She said: "In the interests of effective scrutiny and debate, there needs to be proper opportunity for all members to express themselves robustly and to be heard.
"However, they must do so in a way that is respectful of others.
"Our exchanges are at their best when members strike that balance.
"That will be my expectation of all members throughout the fifth assembly. I will review the record of today's proceedings and make a ruling if I decide it is appropriate to do so."
UKIP group leader Neil Hamilton has sparked a row after describing two senior female AMs as "political concubines" in Carwyn Jones' "harem".